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ZOOLOGICAL SCIENCE 17: 33-45 (2000) @ 2000 ZoologicalSociety

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Furtherlmprovements to the PhotoelectricMethod for

MotileResponses of Chromatophores'Measuring
TakashiYamadat
Ryozo Fujii', andNoriko Oshima
Mtyama, Fbnabashi,
Science,facultyof Science,foho Universi44
Departmentof Biomolecular
Chiba2M-8510, Japan

ABSTRACT-With the intention

of simplitying constructionand operation, improvementshave now been
made to a photoelectric
system formeasuring the motile responses of ch romatophores. 1ntroduction ot chop-

per-stabilized operational amplifiers with a complimentary metal-oxide semiconductor (C-MOS) has

input
broughtabout a much improvedstability of the electronics. Such a featurehas been found to be especially
suitable for measurements requiring higheramplification and longerperiodsof time, e,g., the detection of the

effects of various factorson bright-colored chromatophores. The use of appropriate color filtersthat 1imitthe
spectral range of light used formeasurement has also provento be important, By installinga smal1 filter
close
to the photosensor, we can now record the responses of particulartypesof chromatophores more selec-
tively,while visually monitoring thestates of all kindsof chromatophores innatural color, To minimize the
influence of motile activities of xanthophores and/or erythrophores, the use ot an orange-to-red long-pass

filterisappropriate to optimize recording the melanophore responses. By contrast, the re$ponses of

xanthophores or erythrophores can be recorded more easily by employing a violet-to-blue band-passfilter,
becau$e thatincreases the contrast of images of these cells against the background. Using an orange-red
variety of the medaka Oiyzias, we have also recorded photometrically theresponses ot leucophores, whose

organelles are light-scattering. A long-passfilter was efficient in excluding the intluences of co-existing

xanthophores,

Employing those improvedtechniques, we have actually per-

INTRODUCTION formed many experiments, and some of those results have
Quantitative
and reliableassessment ofcel1ularresponses etal.(1991,
reports by Fujii
been recentlypublishedincluding
isindispensableforphysiologicalstudiesofmotilemechanisms 1993),Hayashiand Fujii (1993)
and others from our labora-

as well as the regulatory systems of effector cells.Among tory.

methods used to record the motile responses of chromato- Meanwhile,severalresearchgroupsworkingonthephysi-
technique has recently gained much ology of chromatophores have frequentlyurged us togivemore
phores,a photoelectric
becauseof itsreliabi1ityand wider applicabi1ity (Fujii,detailed descriptionsabout our techniques than what was
popuIarity
1959; Oshimaand Fujii, 1984; Odman et aL, 1992;cf, also briefly reported intheMethods sections of articles cited above.

Therefore, we have decidedto providethe details of our lat-

Fujii,1993).In fact,that method has greatly contributed to
of the apparatus, along with some suggestions
progressincharacterizing the physiology of chromatophores, est version

and has led to a deeperunderstanding of mechanisms useful forthe actual application of the method tovarious types
involvedincellular motiiity as well as those underlying color ef chromatophores. Itshould be emphasized here thatthis
system may easilybe adapted to laboratory courses in cell
changes ot animals.
While employing that method, we have continued to physiology,because the expenditure required forprepanng
we have
and infact,
improvethe system forbetterapp1icabi1ity, the installation
isrelatively small,

made several signiticant improvementssince we lastpre-

sented a solid description about it{Oshimaand Fujii,

1984). EXPERIMENTAL
+Corresponding
Tel. +8t-03-3480-5891;
author: Material$
FAX. +81-03-3480-5891. 1nthe experiments presentedinthiscommunication, only
E-mail:fujii@biomol.sci.toho-u,ac.jp a few species of freshwater teleostswere employed, butwe
"
Thispaper isdedicatedtothe lateProfessorEmeritus H. Kinositaof
believethatthe method described herecan be applied widely
the University
of Tokyo, who passed away in February,
1999.
tPresentaddress:DiagnosticsResearchLaboratories,Fujirebio,lnc., to many $pecies of teleostsand other groups of animals as
Tokyo 192-O031, Japan,
Komiya-cho,Hachioji, well. The referred to species includes the dark chub Ztacco

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34 R. Fujiietat.

temmincki, the medaka O(yzias tatipes,the platyfish forteleostswhich had the following (in
composition: mM): NaCI,
)(iphophorus maculatus and the swordtail X. helleri,
Adult 125.3:KCI,2.7;CaCI,,1.8;MgC12, 1,B;D-(+)-glucose, 5.6;
specimens were obtained from localdealers, to use
and prior Tris-HCIbuffer, 5.0(pH7.3).The method of irrigating the skin
were keptinfreshwater aquariums forat leasta few days to pieceswith physiological saline or with stimulant solutions
acclimatize. Chromatophoresof the common dendritictype, duringthe measurements was essentially identical to that
namely melanophores, xanthophores, erythrophores and describedelsewhere (Fujii and Miyashita, 1975;Oshima and
leucophores,werethensubjectedtoexaminationoftheirmotile Fujii, 1 984).
responses. A peristalticstroke pump commoniy u$ed inthe labora-
When scales frommedakas, platyfish or swordtails were tory can conveniently introducenormal physiologicai $aline
employed, they were pluckedfrom the anterior, derso-Iateral intotheperfusion chamber at a constant rate {Fig.1).Addi-
partof the trunk. Inthe case of dark chub$, scales trom the tional pumps may be employed to supply other various
darklongitudinal stripe along the middle partof the body were experimental solutions, and theseare especially helpfu1 when
employed.Sometimes,splitfinpreparations(Fujii,1959)were solutions other than the normal saline have to be applied over
used with practically identical results to those obtajned using long periodsof time,UsuaHy however, we applied such
scales from the same species. The excised skin specimens experimentalsolutionsmanuailybymeansofPasteurpipettes.
were immediately immersed ina physiological saline solution Manualapplicationwasespeciallyimportantwhenonlyasmall
amount of a precious solution isavailable. To remove
medjum fromthe perfusing chamber, a vacuum $tream pump
ps cvc operated by tap water is convenient, and when such isnot
available, a sma" drainingpump or an additional stroke pump
W. s may be employed forthispurpose,
"
CFi FGP
Optical system
DCA-EP
ln most studies, transmission microscopes of the ordi-
nary upright type (e.g. OptiphotXT, or Labophot 11;Nikon,
FRA Tokyo)were employed (Fig. 1).
Each scale isaffixedto a coverslip by means of a fine
DOC glassneedle (diameter: ca. O.3mm) thathas been gluedwith
epoxy adhe$ive onto thesurface of the coverslip at bothends.
When a medaka scale isused, itisheld in place with itsbony
ms scale side incontact with the coverslip. For dark chubs and
xiphophorinefishhowever,chromatophoresarepre$entinthe
SE
OL dermis attached to the inner surface of the bony scale (lga
-Qppc IP
r--- and Matsuno, 1980, Fujiiet aL, unpublished observations),
and we thereforeset those scales with the epidermal side in
contactwiththesurfaceofthecoverslip,Whenasplitfinprepa-
MSIE sp EPR
c ration isused, itisheldspread by a pairof glassneedles, as
described by us elsewhere (Fujii, 1959; Fujiiand Novaies,
AC
( - >NDF 1969). The cover$lip with the skin piece on itsunderside is
LS ( >CF2 then mounted ina shallow perfusion chamber, which isthen
FT7.,/t'LYI puton the stage of the microscope formeasurement <Fig, 1),
DCS RM When we employed an invertedmicroscope (IMT-11,
Olympus,Tokyo),a perfusion chamber designedto befunda-
Fig.1. Diagram of the entife system used to measure mentally identical with that described elsewhere was used,
photoelectri-
cally the motile responses of chromatophores. Explanations of because the skin pieces needed to be heldonto the bottomof
inthe figure inthe order fromIeft
abbrevations are arranged to right.
ES: electrical stimulator, AC: commercial alternating current source,
the chamber (Fujii and Novales,1969).Inthat case, the skin
piece isset with its dermal tissuecontaining chromatophores
DCS: stabilized DC power source, PC: perfusionchamber, OP: outlet
up, lnaddition, itshould be noted thatthe topof the perfusion
pipettefor experimental media, MS: micro$cope stage, LS: light
source,PS:photosensor,CFi:colorfilterformeasurement,FRA:field- chamber should be covered with a glassplate. Withoutit, rip-
restrictingaperture,1E:indifferentelectrode,SE:stimulatingelectrode, plinginthe suriace of the perfusing solution causes disturb-
RM: reflectingmjrror, OL: objective lens, FGP: frostedglassplate, C: ances inthe opticai pathway, which results inthe fluctuation
condenser, NDF: neutral densityfilter, CF,: color filter for visual of the recording {Fujii and Novares,1969),
assessment of the effect of spectral restriction,IP: inlet pipetteior The voltages of commercial alternating current sources
experimental media, SP: skin preparation, EP: eye-piece, CVC: cur-
rent-to-voltage converter, DCA: directcurrent amplifier, DCC: DC- tluctuates to a considerable degree especiaily when various
component canceling circuit, LPF: low pass filter, EPR: electronic power-consuming apparati are operating nearby. Suchvaria-
paper-chartrecorder. tionscause fluctuatien$ in the luminosity of the light source

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Measurement ot Chromatophore Response 35

forthemicroscope, necessitating a voltage stabilizing device monly puton theplaneof the intermediate image,i.e. therear
(DCS, Fig. 1), Direct-current voltage stabilizer$ which are tocal planeef the objective lens insidethe eyepiece (FRA,
readily easily available at moderate prices work well forsuch Fig, 1),lnmost cases, we have used a circular aperture with
a purpose, although the wiring to the illuminating lamp should 3.0mm indiameterincombination with a 20-X objective lens.
be alteredto connect with thissupply. Thus, lighttransmittance througha circular area of the skin
with a diameter of 150 pm i$ measured. In many cases, that
To realize higherquality recordings of the chromatophore

responses, the management ot the wavelength of light isalso area issufficient to circumscribe the domain occupied by a
an important factor. For thispurpose,appropriately selected single chromatophore. Ifnecessary however,the measure-
color filters are conveniently employed. What kindof filter is ment ot lighttransmittance can be performed through an area
type of any dimensionand size, and readers can refer to pertinent
appropriate for measuring a certain of chromatophore

wi libe discusses belowinpe rtinentsections, We typically place descriptionsand discussion by Fujiiand Miyashita (1979),Fujii
one or two fi ltersbetween thelight source and thecondenser et aL (1997) and Odman et aL (1992),
lensof the microscope (CF2,Fig,1).Under $uch circum- lfa larger aperture or a Iowerpower objective lens is
through the net effect$ of the responses of a number ot chro-
stances, visual monitoring of thechromatophores employed,
are recorded, and thus, the recordings include a
eye pieces needs be made with the colored light, although matophores

after settingthe celL($) tobe measured at an appropriate posi- higherstatisticalsignifioance(Fujii,1959).Conversely,thea

tion in the microscopic field, continued monitoring isnot can be decreasedby employing an objectivelenswith a higher
is imperative, magnification. For example, the size of the area measured
always necessary. When continued monitoring
to observe the cellsin a monoto- will be decreased by one halfwhen a 40-X objective lensis
itisvery tiringand difficult
insteadof 20-X lens,Alternatively, the area can be
nous, darkfleld. Itsuch observations can be made inordinary employed
made smaller by installing a smaller aperture insidethe eye-
color, they are much easier, and more importantly, additional
information can be obtained about the states of chromato- pieceused forphotemetry,We have preparedapertures of
various appropriate sizes ourselves, although such itemscan
phoreswhose motile activities are intentionally eliminated.
by atso be obtained from machine shops or from manufacturers
Such a condition can be easily realized placingthe
filter
insidethephotographic column of the trinocular system of microscopes by special order. A metal washer fixedto a
inwhich thephotosensorisinstalled (CFi, Fig. 1).Inpractice, holed sheet processed to fitinsidethe eyepiece functions
Similar apertures can be preparedby photographic
we place a small piece of filter just the aperture that
above perfectly.
restricts the area of the skin to be measured (FRA,Fig.1),A procedures. As an example, highcontrast BIW fi1m with a trans-

small broken pieceof a glass filtermay be used for thispur- parentbase(e,g., MinicopyHR 11,FujiPhoto Film,Tokyo)isa
sheet filter A largerpattern of a negative image can be
pose,However,a small pieceof the plastic men- suitable material,

tioned above could more conveniently be employed, because photographedand the processedfilmcan be used conven-
itcan easily be cut intothe desiredsize tofitinside theeye- iently since we can preparean aperture of optionat shape and
size at will (cf. also Fujiiand Miyashita, 1979).
piece(e.g., ca, 12xl2 mm square),
Just above the light-introducing window of the photo-

Photosensing sensor, a finelyground thin glass plate is placed in

order to diffuse lightrays homogeneously tothephotosensing
The photoelectric transducing partof thesystem isfun-
(Oshima chip (FGP, Fig. 1>, This the inadequatetransduction of
damentallysimilar tothatdescribedby us previously avoids
hightintensity owing to the heterogeneousphotosensitivity of
and Fujii,1984>. We have employed a high qualitysilicon pho-
or Sl 226-5BQ; Hamamatsu Photonics, the semiconductor chip insidethesensor.
todiode (Sl226-5BK,
Hamamatsu), buta comparable itemcan be used satisfacto- A mechanical stage adapted to the microscope stage is
rily.The sensor isinstalledinside the photographic column of employed to position the image of chromatophore(s) in the
the trinocularassembly (PS, Fig. 1). When we employ Nikon center of the area to be measured. For accurate positioning

microscopesforexample,atrinocularassemblyoftheTtype of specimens, an eyepiece micrometer with a lattice pattern

was adopted, which enabte$ us to measure motileresponses inscribedcan be conveniently u$ed for thispurpose.
while we the cellsthrough thebinocular
monitor eye-pLeces.

Whichever microscope isselected, iti$recommended thata Electronic circuits

trinocularsystem with such performance be used. Otherwise,
one must employ one of the binocular eye-pieces forthe sen-
by eye.
sor, and the other one to monitor the cells
The photodiodesemployed and described inthisarticle
are of the metal can type, and the cathode terminal iscon-
nected to the metal covering. In order to minimize electrical thispurpose,an operationalamplitier of a new type, namely a
disturbances from the outside therefore,itis recommended
thatthe cathode leadof the itembe connected to the ground
side.
Adiaphragmwithacircularapertureinthecentertscom-
Figure 2 show$ a diagram of the electronic processing
partof the system as itiscurrently employed inour labora-
tory,The firststep partitioned as Part 1 in the figure
isthe
devicewhich converts the changes in the current output of
photosensor the intovoltage changes (CVCin Fig. 1). For
C-MOS chopper-stabilized inputisused (OP,; MAX430, CPA-
typepackage;Maxim lntegrated Products,Sunnyvale,CA).
This item isso designed that the inputvoltage offset isauto-
matically compensated by the bu"t-in chopper-stabilized

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36 R. Fujiiet ai,

Part 1 //'/11a'･p111b
Part2 /11
Part3
HLUT
Rl-Rll :

Rls

oon-irtv C2OP2

R16

Fig.2, Wiringdiagram of the electronic processingpartof the apparatus formeasuring

photoelectric motile responses of chromatophores.
Explanatiens of abbrevations ineach partare arranged inthe order from left to right. Part 1:The podionthatconverts the current output of the
silicon photodiode (SPD)intovoltage. RSi: rotary switch torchanging the sensitivitystepwise, Ri- R": fixedresistorsforfeedback resistance for
OP,.Inorder toachieve a wide range alterationof the current-to-voltage conversion efficiency(sensitivity), those of 5, 1O,20, 50, 1OO,200,500
kQ, 1, 2, 5,and 1O M9 were selected here,OP,: operational amplifier
(MAX430), C,:O.Ol pF capacitor, VRi: 1O-kn variable resi$tor forfiner
adjustment of thg sensitivity, Ri2:4.7 kQ, Part2:The portionthatreve rses the polarity of thevoltage signal and amplifies it,TSi:toggleswitch for
g,ltering the polarity of the signal, Ri3,R": 1O kS2,Ris,Ri6:1OO kQ, C2:O OI
inv" settings are for non-inve rtingand inverting
pF capacitor, OP,:operational amprifier (MAX430).
thesignal. Part3:The portjonthat adjusts the position
The `[non-inv"
and
of the recorder pen,Ri7,Ris'1 1 ,7kQ, Rig,
R2o:12.5k9, R2i, R22:37,5 kQ, R23,R24:62,5 kQ, R2s,R26:125 kn, Rm, R2s:1.00kQ. By employing the above-described values of resistance for

5,b7-,::z'gh8,?g,j"S',a,bbe,r,e･,･g,9･S,a:,b ±
,i,iR.V,e',g,5:ll,,:\J,2g･,,25,,2,M,XS38i,gg,?･,T:,<gLa,,"g,10,:'gg,{･li･,7S,:,:BO
revolution precision
variable resistor (1O
kn),OUT: terminalstoroutput signals to the differential
inputelectronic recorder, between which the
terminalmarked isconnected tothe highimpedanceside inputterminalof the recorder, whiie thatmarked
"H"
"L"
isto the Iovvimpedance side.

mechanism at 400 Hz, inadditionto the desirable feature the signal. Therefore,
of amplify theamplifier pa rtshown as DCA
very high inputimpedance (10"2 9}. Thus, the troublesome inFigure 1 can be abbreviated, except when very highsensi-
offset adjustment thatisinevitably required when using con- tMty isneeded, such as in recording the respon$es of very
ventional operationai amplifiers i6not needed. Furthermore, lightlypigmentedxanthophoresorerythrophores.Inthecase
no extra capacitors andlor resistors are needed forthe offset demonstratedinthe figure,an amplifier with only a row factor
correction.Suchfavorablefeaturesofthedevicearsoprovide
(X1O) was jnstalledwhich was coupled with another optional
a further benefit inthat we need not be concerned about a deviceto reverse thepolarity of the voltage signal. An opera-
possibleincrease ef the offset in a rongrange. Othersimilar tionalampljfierof a similar type to thatused inthe first step
chopper-stabilized operational amplifiers, such as MAX420/ was again employed. By turning a doubie-pole double-throw
421!4221423 <Maxim tntegrated Products>, ICL7652 {Harris,switch (TSi), thepolarity of thesignal can easjly be changed.
Melbourne,FL)or LTCI052 (Linear Technology,Milpitas, CA), We usually record theincreaseinlight transmission, i.e,,the
can also be used, and we have actualiy employed a MAX421 aggregation of chromatosomes in the light-absorbing chro-
successfully, Withoutneed of additional electronic
partsfor matophores, as an upward shift of the trace on the record.
offsetcompensation however,the itemused inthe present Sometimes, one may want to have recordings on which the
study, a MAX 430,isespecially convenient forthose unfamil-
progressof pigmentaggregation bringsabout themovement
iarwith handiingelectroniccjrcuitry. of the pen inthe opposite direction, 1nsuch a case, the device
Ri-Riiare fixedresisto rs forfeedbackresistance of OPi. works conveniently. Actually, the circuit functions as a non-
By turning a rotary switch (RS1), theefficiency of thecurrent- inverting direct current amplifier when the switch isset to the
to-voltageconversion can be altered over a wide range. How- "non-inv"
side. Ifthe switch is turnedto itacts as an
"inv",

ever, the selected resistances denoted in the legend to invertingampiifier. The ratios Ri4Ri3and RielRri4 provide the
Figure2 allow us to make only coarse changes in thesensi- amplificationfactors, When the vaiues of resistance described
tMty,Thus we added a variable resistor, VRi,to attenuate the inthe legendare adopted, the voltage amplification of this
output voltages more tinely in the case
and continuously.
partbecomes 1O.
shown in Fjgure2, thesensitivity of each step can be finely The finalstep (Part 3) of Figure2 isthedeviceforposi-
adjusted from 1OO% down to 30%. tioningthe pen at the desiredposition on the chart betore
Since we employ sufficiently high qualityoperational beginningthe recording. By turning TS2,we can change the
amplifiers thatenable us to execute the current-to-voltage adjustable range of the pen location toan appropriate one. By
conversion very efficiently,there isusually no need to further adopting vaiues of resistance tor Ri7--R26as describedinthe

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Measurementof Chromatophore Response 37

legend,therange can be altered to be 1 .0V, 500, 250,1OO or should be longerthan 1 sec even inthefastest cases. Thus,
50 mV. Itisrecommended thata narrower setting be selected the frequency characteristics of theelectronicdivision isnot
because the adjustment can be performedmore fineiy and required to be so high, and there is no need to employ taster
stably, The actual location of the pen on the recorder is recording devices,such as electromagnetic or cathode-ray
adjusted by turning the precision variable resistor, VR2, oscilloscopes. Therefore, we added high quality capacitors

ln order to simplity the system, we designedthe direct Ci and C2 inthe circuits (Fig.2) which are primarily to avoid
current supply inthispartto be from a common source for undesirable oscillations inthe operational amplifiers and to

drivingthe operational amplifiers,Thus, we determinedthe reduce inevitableartificialdeflections, which occur when RSi

vaiues of resistance of Ri7-R2s assuming the voltage values or TSi isvaried.

of thesource tobe exactly +1 5.0and -1 5.0 V, We
usually use As mentioned earlier, the motile responses of

a handmade power source, in which integratedcircuits sup- xanthophores or of erythrophores giverise to changes inthe
plystabilized +15 V and -15 V. The sanctioned error ot the lighttransmissionwithin rather iimited ranges, and thus,a

commonly available voltage-stabilizing units is±5%. Thus the higheramplitication of thesignal isneeded, Even thoughthe
output voltage of the constructed power supply does not coin- utmo$t care had been taken in constructing and wiring the

cide exactly with the values indicated. Such an error naturally circuits,some noise wa$ inevitably introduced inthissystem.
bringsabout a proportional error in the ranges of adjustable Itissometimes possiblethat electric equipment operating in
values. In practice however,such errors do not giveri$e to or near thelaboratory generatessuch noise, The carefulcon-
serious problemsin the system, because the positioning ot nection to grounding of various components inthe system,
the pen isa relative one. Ifone wants to have more precise such as conductor parts of the microscope, i$usually efiec-
voltage supplies to shift the pen, constant current sources tiveinreducing such trouble.
coupled with precision fixedresistors may be employed, and In thi$way, most problems may usuaily be overcome,
actually, we employed such circuits inearlier versions of the Sometimes however, one might failto reduce the noise,
apparatus. especially when an apparatus thatgenerates vigorous elec-

The signal with the appropriately compensated DC com- tricaldisturbances isoperating. Insuch cases, a low-passfil-

ponent finally leads to an electronic chart recorder with a dif- ter ishelpful,which diminishes noises of higherfrequencies,

terentialinputtype, We use an R-61 or an R-62(Rika Denki, since most noises are of higherfrequencies compared with
Tokyo), or an EPR-10B or an EPR-231A (ToaElectronics, signals due to chromatophore responses. For thispurpose,
Tokyo), butwe believethatmost commercial recorders would we adopted a low pass tilter (LPF)of the second order
be suitabletorthispurpose, Butterworth typethatcan easily be designedto have appro-
The motile responses of chromatophores are relatively priate frequency characteristics (Fig. 3}. In our facility
how-
slow phenomena, when compared with many other faster bio- ever, such a devicehas essentially been unnecessary. We

logicalresponses such as thecontraction ot various muscu- also presumethat in most laboratories, such a device may
latures.1nfact, thetimeconstant of ch romatophore responses not be required under usual conditions. Theretore, thecircuit

1 '
ttRS...---t'
''
'
'R,
,,

'''c,''''''' tt?5?

oo, t't

2tr

''

'st1
5on4os C2Rfi
INR, ''' ';o
OUT
'
',3,q-os.K)

o-v '
3
k
OPi+ +OP2
2os
G

s
R 1

C3

,l;.

Fig,3. Wiringdiagram of the low-passfilter(LPF) of the second order Buttervvorthtype.This deviceisoptional and isused when the noise is
unavoidable. Itisdesignedto be placedbetweenthe above-described interface (Fig, 2) and the electronic chart recorder. Sincethe output of the
interfaceisforadifferentialinputrecorder,adifferentialinputamplifierisappendedasshowninthe1eftpartofthediagram.TheLPFdivisionwas
designedto be able to alterthe cutting frequencyinstepwise fashion.By turning the 3-circuit rotary switch (RS), we can select an appropriate
set. 1nthe circuitdemonstrated, 1e,2,O.5,and O.2 Hz were selected. When we set the RS at the "1",

cuttingfrequencyamong those previously

circuitworks as a voltage follower, and the signals go through itwithout modulation, When the RS ispositionedat or the circuit "2'', "3", "4", "5",

functions as an LPF with the cutting frequencyof 1O,2, O.5or O,2Hz, respectively. To obtain the indicated cutting trequencies,Rs,R6,C! and Cs
have to be selected to have adequate values incombination. The calculated and adopted values shown are as follows: Setting (cutting "2"

"5" O.5,and O.2Hz, respectively}, C2 = 2.2 uF, C3 = 1,1

frequency: 1O Hz),C2 O.2 pF, C3 O.1 pF, and Rs,6 1 12.5k9. Settings
= = = and (2, "3", "4",

pF, while forRs and R6 were calculated to be 56.3,225, and 563 kQ forthethreesettings,respectively.tN:inputterminalstobe connected
to the

output terminalsof the main electronic portion<Fig. 2}, OUT: output terminalsto be connected te the electronic recorder.

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38 R. Fujiiet aL

was housingas an optional device,and

builtin a separate sively used, in which the concentration of Na' ions was
thatdevicecan be placedbetween the above-described in- compensatorilydecreasedsothatthefinalosmolaritywasthe
terfaceand the recorder ifnecessary, Sincetheoutput of the same as a standard saline solution,
interfacewas of the differentiaitypeforthe differential
input Norepinephrine(hydrochioride; racemic modification;
recorder (Fig.2),the LPF should be precededby the differen- Sankyo, Tokyo) was used to inducethe aggregation of pig-
tialinput, For this purpose therefore, a simple differential mentin chromatophores, ltsconcentration isgivenintermsof
inputamplMer was appended, as shown inthe leftpartof the thephysiologicaily active L-(->-isomer. As a sympathetic neu-
diagram. rotransmitter, that amine has been shown to aggregate
The latterhalfof Figure3 isthe LPF proper,inwhich the chromatosomes in light-absorbing ¢ hromatophores via the
cutting frequencycan be altered at 4 steps, The cutting fre- activation of a-ad renoceptors (Fujii,1 993;Fujiiand Miyashita,
quency isdefinedas thefrequency of theinputsignals at which 1975;Fujii and Oshima, 1986), and to disperselight-scatter-
the amplitude of those signalsisdecreased50 %, and at which ingorganelles inleucophoresof some cyprinodont fishvia B-
signal$ at higher frequencies are effectively eliminated. By ad renoceptors {Obika, 1976;lgaetal., 1977;Fujii,1 993).Other
turning the rotary switch with triplecircuits (RS), we can drugs employed duringthe measurements reported jn this
select an appropriate cutting frequencyamong those set study include an a-adrenergic blocker,phentolamine
"1",the circuit works
beforehand,When we set RS at position (mesylate; Ciba-Geigy,Basel),a Ca2'-channel biockerof the
as a voltage follower, and the signals go through itwithout N type, co-conotoxin GVIA (SigmaChemical,St.Louis,MO),
modulation, When we set RS at position endothelin-1 (ET-1 ;human, porcine, etc., Sigma
"2", "3i', "4", "5",
or a mammaiian
the deviceworks as a low pass filter with a cutting frequency Chemical)and a setective antagonist forthe ETB receptor,
of 1O,2,O,5or O.2Hz, respectively. To obtain these fourcut- BQ-788 (Nasalt; Banyu Pharmaceutical, Tokyo).
tingfrequencies, foursets of Rs,R6,C2 and C3 were prepared All tests using skin specimens were carried out at room
with the values neted inthefigu re legend.By employing com- temperaturebetween 20 and 27eC.
monly available capacitors forC2 and C3,appropriate values
of Rs and R6 were calculated, lnour tests,a cutting frequency Appticatiento metanophores
ot O.5 Hz was found to yieldgood results without noticeable Melanophoresare themost easiiy measurable ch romato-
deformation of cellular responses on the recording,Common phoresfortheirresponses, since theycontain very darkly pig-
highperformanceoperational amplifiers may be used as OPi mented organelles (melanosomes) and because the influences
and OP2 (Figure 3). However, we again empioyed MAX430s of chromatophores of other type$ are limited. For practical
as employed in earliersteps of electronic division (Fig.
2), purposestherefore, special color filters may not be required.
However, a $harp-cut filter eiiminating lightraysinthe shorter
Eiectrical stimutatien spectral region (long-pass filter)is useful, especially when
ln some experiments, the sympathetic fiberscontrolling heavilypigmentederythrophores are presentwjthin the area
chromatophores were stimulated by applying pulses to the to be measured. We originally employed an orange-red fiiter
rostral partot isolated piecesof skin. The method was funda- for thispurpose(e.g., O-55, ToshibaGlass,Tokyo; Oshima
mentally identical to thatemployed in earlier studies u$ing and Fujii,
1984).Later, a cotor filterwith a longercutoff wave-
isolated finpieces{Fujii and Novales, 1969;Fujii and Miyashjta, length was used to more effectively eliminate lightraysthat
1975), butmodified somewhat forapplication to scales (Fujii alter theactivities of xanthophores and erythrophore$ {R-61 ,

and Miyashita, 1979).An electronic stimulator {SEN-3201, ToshibaGlass;Hayashi et aL 1996).Most recently, we have
,

Nihon Kohden, Tokyo)was used inthese triais. Inmost meas- used plastic sheet-filters to replace the glassones. Among
urements however,the skin piecewas stimulated ina tieldof such sheets thatare commercially available, triacetate filters
sine-wave alternating current generatedby a CR o$ciliator of FujiPhoto Film<Tokyo), such as SC-54, -56,or -58,have
(AG-203, Kenwood, Tokyo).Such an electrical fieldisknown profitably been employed. In our recent measurements on
to effectively stimulate sympathetic fibers to liberateneu- xanthophores, we generally use an SC-54 filter which elimi-
rotransmitters(FujiiandNovales,1968),Thestimulatingwaves nates lightrays shorter than 540 nm inwavelength. The com-
were monitored on a storage osciltoscope(5t1 1A,Tektronix, parablesharp-cut fitters of ether manufacturers should also
Beaverton,OR), be usefu1 forsuch purposes. Gelatin filters of analogous char-
acteristics, such as those supplied by Eastman Kodak (e.g,,
Chemical stimueation 16,21, or 23A; Rochester,NY) may also be used, although
A K'-richsaline solution was employed to aggregate pig- theyarea1ittlefragilecomparedwiththeplasticonesdescri
mentaryorganellesinlight-absorbingchromatophores,includ- above. Whether the influences of motile activities of these
ing melanophores, xanthophores and erythrophores, and to chromatophores have been successfu"y eliminated can be
disperse light-scattering organelles (leucosomes) in confirmed by viewing the cells throughthe micro$cope with
leucophores, Elevatedconcentrations of K' ions are known to thefiitered light. When ceils to be eliminated of theirmotility
act as a sympathetic stimuius via the release of adrenergic become practically invisible, the trialis certainly successful
transmitterfrom postganglionic fibers(Fujii,1959, 1993;Fujii (Fig. 4B, i).
and Oshima, 1986),Satinecontaining 50 mM K' was exclu- As an example of the recording using the apparatus

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Measurement of Chromatophore Response 39

Fig.4. Photomicrographsshowing the effects of restrictingwavelengths of lightilluminating the skin specimens, (A)Melanophores and
xanthophores ina scale trom a wild-type medaka, Or]Lziaslatipes, as viewed by the standard illumination, employing a natural color balancing
tilter{NCB,Nikon,Tokyo). Being equilibrated in saline containing 50 mM K', melanosomes and xanthosomes are mostly aggregated into
perikaryaot the chromatophores, xl60, (B) The same part of skin as shown inA, but the illuminating lightspecirum is restrjcted by an orange
filter(SC-54, Fuji Photo Film, Tokyo), Melting into the background, xanthophores became practicallyinvisible, The condjtion isfavorablefor
measuring the response of melanophores, (C)The same partof skin as (A), but the light spectrum isrestricted by a violet band-pass filter {BPB-
45);contrast of the images of xanthophores was increased.(D)Erythrophoresina scale froma swordtail, Xiphophorus helieri. Erythrosomes
were aggregated by treating the scale with 2.5 pM norepinephrine. An NCB filterwasused, x320, (E) The same partof the skin as (D), but the
Muminating light spectrum isrestricted by a blue band-pass filter (BPB-50). The contrast of the testaceous part of the erythrophores was

increased,while theircentral yellowishpartsstillremain rather pale and are a shade of green.{F) The same partof the skin as (D), buttine
illuminating Iight
spectrum isre$tricted by a violet band-pass filter (BPB-45). Note that bothtestaceousand yellowish parts of the cells became
dark;thisconditionisfavorableformeasuringtheresponseoftheerythrophores.(G)Two[eucophoresandseveralxanthephoresinascalefrom
an orange-red medaka viewed using the NCB filter, Being equilibrated innormal saline, xanthophore inclusions are dispersed within the cells,

while leucosomes are completely aggregated intothe perikarya,x320, {H) The same partof skin as in{G), butthe light spectrum isrestricted by
a violet band-pass filter <BPB-45). Note thatthe images of xanthophores are much more distinct.Ifthe measurement isperformed inan area
without leucophores, the responses of xanthophore$ can be measured adequately. See also panel
"C"
forreference, (1) The same partot skin as
(G), but the lightspectrum is restricted by an orange (SC-54).
tilter The images of xanthophores became merged into the background,and this
condition isfavorableformeasuring the response of leucophoresselectively.

described in this report, Figure 5 illustrates

the motile nephrine (NE)was A pronounced aggregation
applied. of

responses of a singie melanophore ina scale from a dark melanosomes took place,as seen in the
then quickly right

chub, Zkicco temmincki. In that recording, we can see that partof the recording.
electrical field
stimulationrapidly aggregates melanosomes, Intheskin of bluishdamselfishes, e,g,, the common blue
The inhibitory effect of an N-type Ca2'-channel blocker,co- dam$elfishChrysiptera cyanea, melanophores are closely

conotoxin GVIA, on the action of the fieldstimulationisthen associated with motiLe iridophores(Kasukawa et aL, 1985,

exhibited, and finally,a sufficiently strong solutionof norepi- 1987).The color ot the lightreflected from the iridophores

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40 R. Fujiiet al,

uttttttv-.tt..t'".l.tttttt-I";tt''l.Ittttt1ltttt-..//ttttt-tt/.--F-t.-'+.t'1,+/,:;ttttt:ttt.tttlttt/tttttttt.ttt/''tttt'''1-1t.;'wlt/./ttt:''''`'I'iLt/rLTlt,.tlt

leo '1.1ttttttt.1tt/'''"t't/t'r'//.tttttt't:t.tttt'ttt'1tlttttt'-'/e..;t--/tLtt'ttt'tt.tt.a:,''lf't
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't.t/-Ttt//t.T

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tttt-tt/t.{'':,I･[-E/]/-'l/'ttttt.,L''.//ttt.FFtttttt':'I'ttt'1ttt1'1ttt/tttttt/..,tttt''tttt1'･
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= /-'tt'ttttl1'･1-.,1/,/tttttttt'''///.'''ttt''.1:,tt'-:--1･-a'//'/1t/t/tttl'lt't'11T,1./.utLtt/ttt/t'/t'-1l1':ttt'',,Ltt"
' '/tt･ltt.et.tt.m''tttt...tt.ttt/.e.t
./･･1 tTt'tt/tt/.'!:tttttt.t'tt'''tt
tttt't../ttt' O,3
ttttttt:tt-tttt'
r't

I.Lr-.ttttt. 'ta'- 't''.;ttttt

'4tt/
/
tl''t'rd":11/t'':./,.t/t/t
'tt.'tt'
/
'} '･l-･l',1/ttttt/d,Filtl.l'
Fttttt/tttt'tt.t':ttttt ttttl/ IIt/ti
' '.t:rx.tTtt.ttt
' 5min:': O.4 O.4
o
' i't'"'i ""---k--.-ttht L:.::::LJ
'tl.Lll'-[t.ttt.t''.'r''11"'1/--.]'ttt ['IE'i.,',,t'i;i''r::::''
tttt.di 't. il. l'
1 pMw-CTGVIA
ACu ncu 2,5 pM NE

Fig.5. A typical photoelectric recording showing the responses of a singie melanophore inthe scale from a dark chub, Zlaccotemmincki, along
with indication$ about the physiological treatmentson the bottom, and those of the ordinates on bothsides 1norder tominimize the influence of
metile responses that exist within the area of measurement, the recording was performed using a long-passfilter
of xanthophores
(SC-56}
installedinsidethe eyepiece inthe photomicrographic column of the microscope (CFi, Fig.1).Abscissa,time,the time scale isindicated by a
horizontal bar {5min> on the bottom right partof the figure.The upward shittot thetraceindicates the increaseinlight transmittance, namely the
melanosome-aggregating response of the cell. On the ordinate displayedon the left, the magn:tude of response jsexpressed as a percentageof
the maximal extent of pjgment aggregation attained duringthe course of measurement, takingthe fullydispersedstate as zero. A sudden
decrea$e tn the lighttransmittance inthe right partof the recording isdue to the insertion of a neutrai densityfilter (ND-O.2, FujiPhoto Fllm;
absotoance: O,2,or transmntance=O.63) for 15 sec across the lightpath.Inaddition to the absorption O.2,values of tne absorption or of the
transmission were calculated and graduatedon the ordinate seen on the right. Afterequilibration in
physiologicalsaline, an alternatingcurrent
fieldstimulation (AC; sine wave, 1O Hz, O.7 Vlmm) was applied. A rapid aggregation of melanosomes tookplace,The action of the fieldstimula-
tionwas inhibited by o)-conotoxin GVIA (a]-CTGVIA), but the action of norepinephrine <NE)was not. Discussion concerning thisand relevant
resultswillbe presented ina separate paper.This and thefollowingphotoelectric recordings are al1 unretouched.

may be described as varying between violetand bluish green. contrast of cells against the brighter
backgroundare ve ryuse-
Beingcomplementary, the color of the iridophoresas viewed ful,
Untilrecentiy, we have been using a glassband-passtil-
by transmitted light assumes a greenish yellowto red color ter such B-48S or V-44 (Toshiba
as Glass).The technique
(Kasukawa et al., 1987), ln order to minimize the disturbing comprises the measurement with 1ightrays of thecomplemen-
effects of these iridophores,therefore,a red filter is useful. tarycolor to the spect rai absorption of chromatophores. More
Usually,
we use an SC-60 filter(Fuji
Photo Film)to accurately recently, we have switched to the use of a plastic band-pass
record the responses of such melanophores, filterproduced by FujiPhoto Filmforthispurpose{Fig, 4C, F,
H),Forrecording the responses of erythrophores, a filter with
Applicationto bright-colored
chromatophores a spectral transmission peak of 500 nm (BPB-50) isappropri-
Xanthophoresand erythrophores are rather difficult ate, while one having a peak at 450 nm (BPB-45) is more
objects to assess for theirmotile responses quantitatively, suitable forxanthophores. 1nany case, the use of such tilters
beoausetheircontrastagainstthe1ucentbackgroundinatrans- inevitablyIowers the intensityof Iight arriving at the
missien microscopic fieldi$much smaller than that of mel- photosensor,thus necessitating higheramplification of the
anophores. Particularly when responses of lightlycolored signals.
xanthophores are measured, net changes of lighttransmit- As mentioned above, itis very difficult to assess the
tance are much smaller. A hjgher amplification in the elec- responses of brightly-colored chromatophores photoelectri-
tronic partisnaturally required, and more precautions need to cally, if segments of melanophores are mingled withjn the
be takenduringthe measurement, microscopic field. In such cases, itisimportant to search for
Whenbrightly-ooloredchromatophoresaremixedwiththearea of the skin where no melanophores are present. 1nmany
melanephores, itis practicallyimpossiblete measure their species of teleostean fish,such skin piecescan rather easHy
responses separately.On theother hand,when the melane- be prepared ifone searches fora section where a yellowish
phores are iacking, chromatophore responses can be mea- or reddish tint dominates.Fu rther,itisa ve ry good ideatouse
sured rather easily. in such cases, trials to increasethe brightlycoloredvarietiesoffishspeciesinwhichmelanophor

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Measurement of Chromatophore
Response 41

lost The vanetbes fishselected forourstud- are commonly recordedto record there$ponse
lfone
are genetically of wants

ies, namely, the orange-red vartety of the medaka and red of a single xanthophore, lensof highermagnifi
an ob]ective

xiphophonne fishhave turned out to be very good matenals restncting aperture (FRA,
cation, e g 40X, or a field Fig1)
forstudying the physiological
characteristicsof xanthophores ot a smaller diametercan be employed lnthe recording
and erythrophores, respectively exhibited Figure 6, a 40-× oblective lens was used
in
As an of the applicatton of thismethod
example to First, xanthosome aggregation due to the increased K' con
xanthophores, Figure 6 illustrates the recording of their centration is seen Then, the effect of BQ-788 a selective

responses of them on the scale of a medaka Usuallythe antagonist torendothekn 1 on xanthosome aggregation ts

size of xanthophores is smaller than thatof melanophores exhtbited Finally xanthosome aggregation due to the effect
By recording the transmittance through a circular area 150 of NE solutton ts displayed

pm tn diameter therefore, the responses of several ot them An example of the responses of seve ral erythrophores in

'lk"g
ikl
,l",gtl,,'iL'1
J`trl' j'ii
i' i } jyFtF
100 I
]Vl ll
"

*OvE=89R
s;l,}rEgsi'
l IIl:3 lIht l
'lilI jl

t･l ,
dIg
'i:
ftl'

so{gR"m. 1urts
Llne
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iv

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t-ti
ll,l
': lr
I?

IlrvlT.tlt
'l'

re o w,4l
-
- lt

50 mM
Ll
K+
-
2SnMET1
25 nM BQ788

Fig 6 A typtcal recording showing the responses of a few xanthophores in a scale from a medaka Oryztaslampes ot the orange red vanety
lnthisparticular recording, a band pass filterwith a spectral transmlttance peakat 440 nm (V44 Toshiba Glass)was employed iorincreaslng
the contrast of the images of the xanthophores against the background The upward shlftot the trace tndlcates an increase in 1ight transmittance,
namely thexanthosome-aggregating respon$e of the cells Afterequ"ibration in physiological saline K' rlch saline was applied to test the normal
responsiveness of the cells Following the treatment with a selectFve blockertorthe mammalian ETB receptor BQ 788 mammalian ET 1 was
applied Finally2 5 pM norepinephnne (NE> solution was added to lnduce a maximal level of pigment aggregation

1OO

A*v=9ttoyo?50vEa2-s-

2UtPA -tMPA
Fig 7 A typicalrecording showing the few denervatederythrophores in a scale from a platytishX)phophorus maculatus
responses of a ln
order to increase the contrast of the images of the erythrophores agatnst the backg round a band passfLlter with a spectral transmtttance peak
at 450 nm (BPB45) was employed The upward shift of the traceindicates an increase in the 1ighttransmittancenamely the erythrosome
aggregating response of the cells Afterequilibration in physiological saline, the concentration of K' in the medlum was increased to 50 mM A
moderate aggregation of erythrosomes took placeAfter equilibratton again in physiological saline the scale was treated with an a adrenergic
blockingagent phentolamine(PA}for2 mtn Then K' nch sa[ine containing the same concentratson of PA was applied The erythrosome
aggregatlon wa$ not inhibited Acetylcholine was toundnot tohave any erythrosome aggregating action Norepmephnne {NE)was very effechve
tn inducing a maximal leve[of pigment aggregation After equilibration in physiologtcal saline again, the action of NE was testedtn the presence
of PA The action was totally blocked The tneffectiveness of PA on reaction to an increased K' concentration was again confirmed Afterthe
app"catlon of NE again atroptne (Atr) was applted and iteffecttvely dispersedthe pigment The details of these results and discussion relevant

to this recording will be presented elsewhere

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42 R. Fujiietal.

a platyfish
scale of a isshown in Figure7, A K'-rich saline inthe dermisof the kMifish Flindulus
melanophores, such as
moderately erythrosomes, but acetyolcholine
aggregated (Menter of the medaka Or)czi'as (Fujii
et al,, 1979) and
and
(ACh)did not. By contast, NE strongiy aggregated the pig- Miyashita, 1979;Obika,1996),lnsuch cases, itispractically
ment. An a-adrenergic biocker,phentolamine(PA), had no impossibleto measure Ieucophore responses $etectively.
influenceon the action of K' ions. Occasionally however,a leucophore can be found by itself,
i.e.without an overlying melanophore and fortransmission
Applicationto cyanophores photometry,such a leucophore ispertectly .
Motileresponses ot blue chromatophores <cyanophores) Physiologicalstudies of ieucophorescan be performed
found insome callionymid fishcan rather easily be quantified very efficiently using individuals of the orange-red variety of
by thephotoelectric method, becausethe contrast of the cells medaka, becauseintertering melanophores are lackingintheir
against the background isfairly high(Godaand Fujii, 1995), skin. Inaddition, they are more easiiy availablecommercially
The ve ry same equipment used inthestudy of melanophores now than are wild-type specimens. Again,a red Iong-pass
can be employed torecord cyanophore re$ponses, and Goda filteris very usetul formeasuring the motile responses of
and Fujii (1995)actually recorded the responses of leucophores,because the influenceof motile activities of
cyanophores and reported a recording, A red or an orange- xanthophores can be eliminated, as was demonstrated inFig-
to-red filter should be employed to eliminate the activities of ure 41 when an orange-red filter (SC-54, Fuji Photo Film) was
xanthophores or erythrophores, such as when the melano- used, ln thephotoelectric recording displayed in Figure8, a
phores are studied. more reddish fjlter(SC-56, Fu]iPhoto Film) was used inorder
to record the responses of individual leucophoreson scales
Applicationto leucophores more exactly. An elevation ot the K' concentration intheper-
When we wanted to describethe responses of ieuco- fusingmedium gaverise tothedispersion of leucosomes,and
phores, we have conveniently employed an industriai micro- NE exhibited thesame effect.
scope installed with dark-field epi-illumination optics (FuJiiand Being different from the cases of other chromatophore
Miyashita, 1979; Fujiiet aL, 1997),However, such a micro- species (cf. Figs.5-7),leucosomesbecome aggregated when
scope israther infrequently foundinbiological iaboratories. 1n theskin preparation isequiribrated inphysiological saline. Upon
this study, therefore, we decided to demonstratethat by stimulation by an increase in K' concentration, NE, and so on,
employing an ordinary transmissionlightmicroscope, theleucosomes disperse. 1nthe normal system mode thatjs
Ieucophoreresponses can be photoelectrically measured, commonly employed forrecording theresponses of other types
Throughatransmissionmicroscope,leucophoresappear of chromatophores, ieucephoreresponses to these agents
as darkcells like melanophores, and one may frequently mis- are recorded as downward shifts of the trace,as exhibited in
take them formelanophores. This isbecause lightrays are Figure 8.Reversingthe polarity of the electrical signals tothe
strongiy scattered away from the light path by the light-scat- recorder aliows the generationof recordings in which the
teringorganelies (leucosomes) within the cells, Leucophores leucosome dispersion results in an upward shift of the trace.
are commonly foundtobe locatedconcentricaliyjust beneath In the electronic system presented here, we can easily

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tli/.I.1/; ttt.tt't.sf
･-,

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/li/:,: 'l
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".,c

er;iiiflii,F' irllll,i,,
, .
r7X･

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ll,'rl
'I
''
/: '
i"i'
i:
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I ,,tt-, 1'1.-ttl/.tt /t'di.l:

lii,ml
,iua11i
li:ii.i.ig'lii･l-l･,,1'
;1.1la,1'..l･ali'
,1,
:･ ' i'

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soegg" ･;l-･-l '
I1･15.ge
-i ･i
'I

±
1,,
.tttk,./t '.,,'l
i't'

min ';'i
pt ttt
;.e･
･l/･l"'}i
f't
..r
-k
i,i. '/l.･l'
1'
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'/g,'
..l
i,1'
'. ..t-
l, tl･
'll･:}i

-;eljll i'i'
1 ･/t'.:l
'
ttt
i･'l
i-Tt'i;ITIII,..;'l..fi
"-･li -'me'Tr'vt--g
-
t.I･
tllr' ':i::-' il･'
v!'if,
li.- 'T"r l
100
.4 .Ir'.l,ll･'i " :-
i
50 mM K+ 2.5 NE uM
- -

Fig, 8. A typical recording showing the responses of a single leucophoreina scale pluckedfrom a medaka of theorange-redvariety. 1norder
to minimize influences oi motile responses of xanthophores {SC-56)
existingnearby, a red long-pass tilter was employed. The normal mode for
recording the responses of ordinary chromatophores was used. Thus, the downward shifts of the trace indicates the extent of leucosome
dispersion. An increase tn K' coneentration and NE dispe rsed the leucosomes.Momentary downward shiftsfrom thetraceare due to decreases
in light transmntance caused by small air bubb[es that passed across the microscopic field being measured.

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Measurement of Chromatophore
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reverse the polarityot the signal just

by turning the toggle frequentlyemployed to describethe responses of chromato-
switch (TSOin Part 2 of Figure2, ln such recordings, the phores of other groups of animals as well, However,
many

responses of the cells can then be graduatedas an upward that method has occasionallybeen criticized foritsrather low
transitionof the trace. quantitative nature. Direct microscopic measurement of the
diameters of indMdualchromatophores or the lengthsof cel-

Further quantification of responses lular projectionshassometimes been tried (e.g.,

Spaeth,1916;
1n recording the responses ot dendritic chromatophores Kinosita,1963).However, since itemploys an eyepiece
photoelectrically, the magnitude of response is usually ex- micrometer, that procedureisrather troublesome.Photomi-
pressed interms of the percentage change recorded duringa crography i$the $implest way ina sense, and can provide
certain serie$ of measurements. Ifone wants to describethe visua)records of higher However,thatmethod cannot
quality,
response by light transmittance or by absorbance, a neutral follow rapid responses of chromatophores, and results must

filterof relatively low densitycan conveniently be utilized, be presentedwith only a limited number of images. The
lnthe final partof the recording shown in Figure 5 that microscopic images can be recorded on videotape (Fujii et

exhibitsthe responses of a melanophore from a darkchub, aL, 1 991}, butthismore convenient method stil1suffers froma

for example, a sudden and brietdownward deflection of the lack of resolving power,and selection of images isnaturally
traceis $een during when melanosomes were completely needed forpresentation such as with photomicrography.

aggregated by theapplicationof 2.5 pM NE. Thiswas due to Meanwhile, the photometricmethod which measures

the inSertion of a neutral densityfilter with an absorbanee of transmittance throughthe skin has become a convenient
O.2(ND-O.2, FujiPhoto Film)for 15 sec across the light path- method tocharacterize physiological responses of chromato-

way, between the illuminator and the condenser lensof the phores.Ifcompared with the above-mentioned methods,

microscope, When we take themaximal aggregation of pig- unfortunately the measurement of light intensity through a
ment to be the nuIl absoubance, or the unit transmittance there- restricted area of a microscopic fieldnaturally involves rather

fore, the levelattained equals O.2 inabsorbance, or O.63 in complicated procedures,which frequently leads to subopti-

transmittance, 1nthisway, the magnitude of the response can mal application. With the intent ot making this method more

be transformedinto absorbance or intotransmittance, as availableto researchers inthe field of pigmentcell physiology

indicated on the righthand ordinate, forexample. therefo re, we have kept mindful of simplifying thesystem, and

When changes inthe transmittance due to the motility of have publishedimproved methods at occasional stages of
chromatophores are smailer, such as when the responses of development<Fujii, 1959; Fujii and Novales,1969;Fujii and

xanthophores or of erythrophores are studied, a lighterneu- Miyashita, 1975, 1979; Oshima and Fujii, 1984; Oshima et
traldensity filter
may be appropriate. Forexarnple, we occa- al., 1984). In the present communication, we have now

sionatlyuse an ND-O.1filter(absorbance:
O.1 FujiPhoto Film)
,
describedthe latest version of our system.

to measure the responses of Oryzias xanthophores and Some workers are stillusing photoconductive
elements,
)(iphophoruserythrophores, represented by cadmium sulfide (CdS) cells, as the photo-

We have just indicatedabove that the responses of sensors for thefaint light through the microscope. However,
Ieucophoresmay be recorded by expressing the decreasein we highly recommend the use of silicon photodiodes,the
lighttransmittance as an upward shiftof thepen. In batteries", which have highpertormanceand
t"solar
such case$ so-called
al$o, a neutral filter can be employed, After confirming the which we have been using inrecent years.Withouttheneed
aggregation of leucosomes intothe perikayon, a neural den- foradditional curFent which can complicate the electronics,
sity filteris insertedacross the lightpath.The resultant the silicon photodiodeitself generates photo-current in pro-

decrea$e inlight transmittance causes an upward transloca- portion to the amount of photons itreceives. Inaddition, sili-

tionof the trace, con photodiodeshave similar or superior spectral sensitivity

ln eithercase, i.e,when the insertion of a densityfilter characteristics than do human eyes and many other animal
causes adownward or an upward shitt of the trace, the gradu- specles,
ation of the ordinate can be calibratedinto the tran$mittance For accurate and stable conversion of thephoto-current
or intotheabsorbance, whichever ispreferred. 1nusual physi- tovoltage, integrated circuitsof a new type, i.e.C-MOS-input,
ological or pharmacological analyses of responses, however, chopper-stabilized operational amplitier$, have now been

such a transformation of graduation on theordinate isnot an introduced. Without requiring any additional electronic parts,
indispensablecondition. these itemscan cancel the input offset by their built-in cir-
cuits. Consequently, ve ry good1inearity inthe current-to-volt-
age conversion as well as in the voltage amplification is
DISCUSSION
achieved which allows us to obtain very reliable results. In
To date, severai methods have been employed for any case, the simplification and detailsof the electroniccir-
describing the responses of chromatophores, Among them, cuitsshould helpnovices to construct theequipment and wir-
thewell known melanophore index (M.l., or Ml) which was ingof allthe stages. Actually, no adj ustment of theapparatus

originally described forassessing the responses ot amphib- isneeded ifcorrectly wired. When we have used conventional

ian melanophores (Hogbenand Slome, 1931} has been operational amplifierswithout the built-in compensating com-

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44 R. Fujii
et aL

partment, thelong-term drift

of the offset has always been an phores and xanthophores (cf.
Yamamoto, 1975). Similarly,
annoying and inevitable problem,That defecthas now been scale$ from the red skin of xiphophorine fish,
e,g, platyiish
corrected, and the system can now be used without caring and swordtails (Matsumoto, 1965),and theventral skin of the
about the increasing offset, neon tetra (Hayashi
et aL, 1993) are good materials forthe
Usually,
chromatophores of several typesco-exist inthe study of erythrophores, because other types of chromato-
skin. Even when the area of skin to be measured forlight phoresare absent there.Such color mutants and skin speci-
transmittance i$restricted toa size as small as a single chro- mens inwhich the targeted
typeof chromatophore issuitably
matophore, partsof other types of chromatophore are distributed shouid provideuseful materials forfurther studies
commonly found within the area of measurement, Unless pre- on the physiology of chromatophores.
cautions are taken to exclude the influence of the motile Employingequipmentverysimilartothatdescribedhere,
activities of other chromatophores, the responses of thetar- butusing operational amplifiers of earlier types and glass color
getedcell{s) are inaccurately recorded, As describedindetail filters
placed between the illuminator and the condenser lens,
inthisarticle,management of thespectrum of illuminating light we had already performed studies on the physiology of mel-
using an appropriate coior filterissimple butquiteuseful. Mel- anophores ot various species of fish(Fujii et aL 1993;Hayashj
,

anophores are easy objects for photometry,and their and Fujii, 1993,1994},and of erythrophores inthe abdominal
responses may be observed even without an additional color skin of the neon tetra(Hayashi et aL, 1993). Employing
filter,
aithough we recommend the use of an orange-red or a exactly the same apparati as those descrjbedinthe present
red sharp cut filter,
By contrast, measurement of the responses artjcle, we have recently studied the motile responses to
of other type$of chromatophores isa considerably more diffi- endothelins of xanthophores and erythrophores of some
cuit task forthe photometric method, and the use of a proper teleosts forthe fir$t
time (Murata and Fujii, 1995>. Working on
color filter isindispensable. In the presentarticle, we have the newly found bluechromatophore$, termed cyanophores,
discussedtheirappropriateuse,althoughonlyafewexamplesof callionymid fish,we have aiso employed thesame system
were presented. We know that the colors of chromatophores (Godaand Fujii,1995). Sincethen, we have been using the
can vary subtly delicately,depending on the species, the system describing here,and have enjoyed the much simpli-
location on the body,the dietary conditions, and so on. Thu$ fiedoperation and the increasedstability,The usefulness of
worke rs should takespecial care to seek appropriate filters to thepresentphotometric method has now been assured, and
be used insuch instances. we are able to record the moti1e responses ofchromatophores
Unlessthe investigation isspecificallydirected to a cer- of various types.The reliable results thusobtained willsurely
tainspecies of animal, experiments on metanophores can afford useful information for a deeper understanding of the
usually be performedwithout selecting a specie$ of animals, physiology pigmentary
of effector in
cells general,
becausemelanophores existaimost ubiquitously, lnthe case Opticaland electronic partsemployed formanufacturjng
of other type$ of chromatophores, by contrast, selection of thisequipment can now be obtained at reasonabte prices,
materials is fairlyimportant, although we natura"y work on and once con$tructed, they can be operated very ea$ily. Thus,
definedspecies where the study isprimarily concerned with a similar photoelectric system may be adopted even inpracti-
thatspecies, Fortunately, we can now obtain variously col- cal courses of animal physiology wherein undergraduates learn
ored animals frompetshops and aquarists rather easily. Thus, the physiology and pharmacologyof chromatophores ot vari-
we have successfully employed various beautifully colored ous animals. We believe that the pigmentaryeffectorsystem
aquarium species, eitherfrom inland wate rs or fromcoral reefs that exists inthe skin of these animals provide an excellent
(cf. Fujii, 1993),which are ot course wild types. approach forstudents to understand the mechanisms of cel-
In some cases however, color mutants are convenient lularmotility as well as those underlying the nervous and
materials to study chromatophores. Forexample, the medaka endocrine controlof cells.
or the Japanese kil1ifish, Oty:zias latipes,isa small freshwater
teleostean species, which iswidely employed invarious fields
of investigation, Scalesexcised from specimens of the wild ACKNOWLEDGMENTS
type(gene symbol: BF?)providegood material forthe study of We thank Dr.M. Sugimoto,Mr. N. Mu rata and MissA. Masagaki
melanophores. Anothermutant, called `Lblue" (BO, thatiacks fortheirinterest and skilled as$istance. This work was partlysup-
xanthophores may also be employed forsuch studies. By portedbygrantstoR.F.fromtheMinistryofEducation,Scienc
Cultureof Japan.
contrast, (bR), and
"orange-red"
specimens which are now more
readily available than wild ones are very convenient materials
forstudying xanthophores, because melanophores are miss-
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