PHYSIOLOGY AND PHARMACOLOGY
OF COLOR
CHANGE IN THE SAND FLOUNDER
SCOPTHALAMUS
AQUOSUS
George T. Scott
Department
The Marine
of Biology,
Biological
Oberlin
and
Laboratory,
College,
Woods
Oberlin,
Hole,
Ohio
Massachusetts
ABSTRACI-
An elucidation
of the mechanisms of chromatophore
control in tclcost fishes involves
consideration
of the participation
of the nervous system (pigment
concentrating
and dispersing fibers) and an evaluation of influences mediated by pituitary
hormones.
The sand
flounder,
Scopthalamus
aquas-us Norman (Lophosetta
muculatu Gill) was investigated
by
neurological
and pharmacological
methods.
Nerve cutting, blocking
and stimulation
experiments
revealed unequivocally
functional
pigment aggregating but not dispersing fibers.
Acetyl choline and eserine when injected into the spinal cord bring about excitation
of
pigment concentrating
nerve fibers but have no local effect on melanocytes.
Drugs causing
local pigment aggregation are aromatic ethyl amines or hydrazines.
Epinephrine,
norepinephrine ‘and dopamine are extremely
active.
The most potent pigment dispersing
drugs
were certain phenothiazine
tranquilizers.
Pretreatment
with pyrogallol
markedly potentiated
epinephrine
and norepinephrine
but
raised the effective dose of five of the drugs having pigment dispersing properties.
The pharmacological
data suggest a catechol amine as the transmitter
at concentrating
nerve fiber endings, and that the enzyme catechol-o-methyl
transferase is involved in the
physiology of color change in the sand flounder.
INTRODUCTION
Integumentary
color changes of animals
represent some of the most dramatic examples of adaptation to environment,
The
assertion of Briiche in 1852 that color change
in the chameleon is controlled by nerves
and the speculation of Paul Bert in 1875
that two sets of fibers are involved, set the
stage for a vast field of research to come
somewhat later. Active investigation
was
stimulated by the studies of Alfred Redfield
on the horned toad Phrynosoma. His research established the fact that the contraction of melanin pigment during nervous
excitment, initiated by a noxious stimulus,
was brought about by the cooperation of
nervous impulses delivered to the pigment
1 The author wishes to express appreciation
to
my former students, James C. Hickman and Paul
S. Treuhaft, for able assistance in carrying out the
experiments.
This investigation
was supported in
whole by Public Health Service Research Grant
MH 03903, from the National Institution
of Mental
Health to Oberlin College.
cells by the sympathetic nervous system
and the secretion of adrenin by the adrenal
gtands (Redfield 1916, 1918).
Research in chromatophore
physiology
has been carried out on lampreys, elasmobranchs, teleosts, amphibia, and reptiles,
but the bulk of it has involved the fishes.
Several important reviews have appeared
(Waring 1942, 1963; Parker 1943, 1948;
Brown 1962; Fingerman 1963, 1965).
The leading investigator for many years,
G. H. Parker, divided animals into three
groups depending on the degree to which
direct innervation of chromatophores occurs.
Control entirely independent of neural activity ( aneuronic) is found in certain forms
in which the response is regulated solely by
the activity of blood borne hormones. Mononeuronic chromatophores are innervated by
a single set of motor nerves, the excitation
of which always results in aggregation of
pigment.
Dineuronic
chromatophores, on
the other hand, are innervated by separate
pigment concentrating and dispersing fibers.
R230
COLOR
CIIAN’X
IN TIIE
FiShC3
All three types arc claimed to be found
among the fishes, with the possibility in
some forms of a combination of hormonaland neural effects. A precise definition of
relevant neural and hormonal influences.
may be difficult.
Regulation of chromatophores strictly by
hormones carried by the blood is probably
the archaic system; neural control where it
exists has subsequently been superimposed
on it, The melanocyte stimulating hormone
(MSH) elaborated by the posterior or intermediatc lobe of the pituitary has been
found to be a potent pigment dispersing
agent in certain animals.
Investigations
on elasmobranch
fishes
show no evidence for direct neural control
of melanophores as hypophysectomy renders
the animals permanently in the pale condition and injection of posterior lobe pituitary extracts or intcrmedin causes dispersion of pigment. Adaptation to light background may be due simply to the removal
of MSH from the blood (Lundstrum
and
Bard 1932), control therefore being unihormonal ( Abramowitz 1939 ) . Assertions have
been made, however, that blanching in
some elasmobranchs may result, at least in
part, from the release of another pituitary
hormone (Waring 1938, 1963). In either
situation, melanophores are assumed to be
aneuronic.
Mononeuronic control, however, was reported by Parker and Porter (1934) in the
smooth dogfish Rrluste&~ c&z
Evidence
was based on the observation that incisions
in the pectoral fins give rise to light transitory bands or splotches distal to the cuts that
persisted in some cases for several days.
They concluded that melanin aggregation
was caused by the release of a mediator
substance from the nerve fibers which had
been stimulated by cutting, Electrical stimulation of the fins had the same blanching
effect as did cutting. Parker (1943) further
reported that he obtained a substance by
extracting fins of Mustelus which, when
injected into dark dogfish, would cause
melanin concentration in the vicinity of the
SAND
FLOUNDER
R231
injection. This substance, soluble in oil, was
called selachinc.
In the study of teleost fishes, agreement
is unanimous that melanophores are innervated by pigment aggregating neurones.
Electrical stimulation of the nerve fibers
causes concentration
of pigment; cutting
the fibers results in dispersion. In addition,
a majority of investigators have concluded
that tcleosts may possess a second set of
nerve fibers which, when excited, cause
active pigment dispersion.
Evidence for
thcsc dispersing fibers derives from observations resulting from sectioning of spinal
nerve fibers, principally
in the fins of
fish adapted to a white background. The
operation typically results in a dark band,
which fades in a few days, distal to the
cut in the area innervated by the fibers.
If then a second cut is made parallel to the
first cut and somewhat distal to it, the
faded band will repeat its transient darkening. Prolonged stimulation of the nerves by
injury at the site of cutting is the interpretation offered for this reaction. Parker
( 1936) claimed he could block the conduction of these cut fibers by the application
of a cold block that would prevent pigment
dispersion distal to the block.
Interpretations
suggesting the presence
of dispersing nerve fibers and hence dual
innervation
are reported in investigations
on the following tcleosts: the killifish, Fundubs
(Mills 1932; Parker 1941b) ; the squirrelfish, Holocentrus (Parker 1937); the paradise fish, Macropodus (Kamada 1937); the
catfish, Parasilurus ( Matsushita 1938 ) ; the
bullhead, Ameiurus
( Parker 1940) ; the
goby, Gobius (Fries 1942); the minnow,
Phoxinus (Giersberg 1930; von Gelei 1942;
Gray 1956 ) ; the angelfish, Pterophyllum
(Tomita. 1940); the goby, Chasmichthys
( Fujii 1959) ; the crucian carp, Carassius
(Iwata, Watanabe, and Kuihara 1959).
Opinion has not been unanimous, however, supporting the validity of Parker’s interpretation that pigment dispersion resulting from nerve sectioning is caused by continued injury discharge in dispersing (but
not aggregating) fiber stumps (Waring 1942,
1963; Healey 1954; Gray 1956; Young 1962).
R232
GEORGE
Osborn ( 1939 ) , Waring ( 1942 ) , and Healey
( 1954) discussed the possible interaction
of blood hormones on dencrvated melanophores. An alternative explanation offered
by Gray ( 1956) is based on the assumption
that the removal of central nervous control
might result in some inherent dispersing
mechanism in the melanocyte coming into
play and that later the melanophores become hypersensitive to diffusing aggregating ncurohumors. Of interest and pertinent
to this last possibility is the observation
that, sometime after nerves have been sectioned, mammalian
autonomic
effecters
have a lower threshold to chemical stimulating agents ( Cannon and Rosenbleu th
1937). Furthermore,
both Smith ( 1941)
and Parker (1942) reported hcightcned
sensitivity of fish melanophores to adrcnin
in dencrvated areas.
It is possible, therefore, that the appcarante of and later fading of a dark band following nerve sectioning, or the appearance
of a secondary band after recutting a faded
primary band (Parker effect), may have
other interpretations
than that originally
proposed by Parker and subsequently supported by several investigators.
The dominant control mechanism in teleosts is certainly nervous, although in some
forms, neural activity may operate against a
hormonal background. In Ameiurus, MSH
appears to supplement pigment dispersion
initiated by nerves ( Abramowitz
1936a;
Parker 1940). A similar relationship is reported in the eel AnguiZZa (Parker 1943).
The killifish Fundulus, on which a vast
amount of work has been done, shows typical color changes after hypophysectomy.
Extracts of Fundulus pituitaries will, however, darken a blanched, denervated portion of the caudal fin, showing that when
the melanophores are released from nervous
control, they will respond to MSII.
Its effect may normally be dominated by a
neural mechanism except perhaps in cxtreme conditions of dark background adaptation maintained over long periods of time
( Kleinholz 1935 ) .
Additional
evidence that pituitary hormones are not significantly
involved in
T. SCOTT
melanophore pigment dispersion was obtained by Weisel ( 1950) on several teleosts
which were observed to be refractory to
pituitary extract injections. His investigations included the mosquito fish, Gambusia
affinis; the California
killifish,
Fundulus
parvipinnis; the opaleye, GireZZa nigricans;
the mudsucker, Gillichthys
mirabilis; the
green sunfish, Lepomis cyanellus, and the
grunion, Leuresthes ten&s. Gray ( 1956)
reported a similar nonparticipation
of pituitary hormones in the chromatics of Phoxinus
phoxinus.
Accelerated interest in hormonal agents
that would produce color changes in animals
followed the discovery and identification
of
adrenin by Redfield (1916, 1918) as the normal physiological agent causing blanching
in the horned toad under conditions of excitement. Subsequently many papers have
been published relating to the possible role
of epinephrine in fish chromatophore physiology. Marc recent publications emphasize
the potent pigment concentrating effect of
the hormone. Evidence that concentrating
nerve fibers secrete a neuro-hormone transmitter was reported by Parker (1934, 1948),
Abramowitz
( 1936b), Dalton and Goodrich ( 1937), and Matsushita ( 1938). No
conclusive finding identifying the transmitter at the chromate-neural
junction has
been obtained. The pharmacological
approach of Spaeth and Barbour ( 1917),
Wyman ( 1924)) Gilson ( 1926) and Veil
( 1936) suggests that an epinephrine-like
compound is involved. Fujii ( 1961)) using
the adrenergic blocking agent dibenamine,
concluded that epinephrine is the transmitter in the gosby Chasmichthys gulosus.
Flatfish
The flatfish, on which the first observations of fish chromatics were made, show
some of the most striking examples of adaptation to background. Pouchet (1876) demonstrated that sectioning
of peripheral
nerves in the European turbot led to darkening in denervated areas and that electrical stimulation of spinal nerves resulted in
pallor in the area supplied. Sumner and
Keys ( 1929) demonstrated that the chain
COLOR
CHANGE
IN
THE
SAND
FLOUNDER
R233
of events resulting in color change in the fecting the physiological functioning of the
Mediterranean turbot was initiated in the junction or the cell.
eye in response to the ratio of reflected to
The sand flounder Scopthalamus aquosus
incident light falling on the retina. Of appeared to be promising biological material
particular interest was the response of the for this research because of a neural comturbot Rhomboidichthys to illuminated var- ponent in the control of its chromatics and
iegatcd background, in that the resulting
because of their availability in ample quanskin patterns in adaptation depended not , tity at Woods Hole (as a by-product of squid
only on the relative amounts of black and collecting).
It soon became clear, howwhite in the background but upon the ever, that more research on the mechanism
degree of subdivision (Sumner 1911). Mast
of chromatophore control in this fish was re(1916) described a similar capacity for
quircd before meaningful pharmacological
adaptation to a contrasted pattern in the
experiments could be carried out.
fluke Paralichthys.
When vision was preExperiments were carried out to explore
vented by blinding (Osborn 1939), the fish
further
the nature of nervous control of
assumed a medium uniform shade supportchromatophores and to evaluate the effect
ing the assumption that “pattern spotting”
on melanocytcs of a large number of drugs
was initiated in the retina.
possessing neuro-physiological
activity. The
The common “sand flounder,” “sand dab,”
following
is
a
report
of
the
investigation.
or “window pane,” Scopthalamus aquosus
was included by Osborn ( 1939) in his work
Methods
on the flatfish. He carried out cxpcrimcnts
The
fishes
were
taken by otter trawl in
in which he cut sympathetic nerve chains
Vineyard or Nantucket Sound and were kept
and spinal nerves and reported dispersion
in large stock tanks of neutral tint in the labof melanocytes in the dcnervatcd areas of
ortitory. Most of the fish used in the experithe skin; also noted was the refractoriness of ments were conditioned in these tanks for
melanophores to injected pituitary extracts.
about a week. Aquaria for background adFive years ago, the author developed an aptation were arranged by placing wooden
interest in research on a form with neurally
pens with sides 8 inches (20.3 cm) high in sea
controlled chromatophorcs that would be tables. Black or white ceramic tile were
suitable for pharmacological
investigation.
placed in the pens to form a bottom and
Previous work had indicated that certain
were extended up the sides 6 inches (15.2
recent psychotherapeutic
drugs markedly
cm). Each pen had an area of approxiinfluenced the melanocyte control system of mately 2 square feet (0.18 m2) and could
the frog. The ataraxics or tranquilizers
accommodate two or three fish weighing
evidently affected brain centers controlling
between 4 and 16 oz (113 and 454 g).
rclcase of MSH, resulting in darkening of Running seawater at 19 to 20C was mainthe animal (Scott and Nading 1961). The tained at a depth of 5 inches ( 12.7 cm).
psychic energizers, on the other hand, acted Illumination
was provided by two 40-w
antagonistically,
causing suppression
of fluorescent lamps at a distance of 3 feet
(1 m) above the sea table. Occasionally a
darkening (Scott 1962). To explore the
basic activities of these agents, it was desir- fish was found to be a poor adapter and
able to extend the investigation
to a pe- was discarded as experimental material. Alaquosus possesses
ripheral neurally operated mechanism ob- though Scopthalamus
viating the complexity of the central ncrchromatophores containing pigment other
vous system. The chromate-neural junction
than melanin, adaptation to background
was of special interest because of the pre- principally
involves reactions of melanosumed presence of a neuro-secretory mcchaphores; the observations reported are therenism acting on a target cell that could serve fore primarily
responses of this type of
as a built-in color indicator J of factors af- pigment cell. No attention was given to the
13234
GEORGE
T. SCOTT
COLORCHANGE
IN THE
possible differentiation
in responses of the
other chromatophores.
Neurological operations or drug injections
were performed either directly in the pens
or in a large black photographic developing pan provided with running seawater.
Photography of the fish was done in the
pan, using a Kodak Retina III 3.5mm camera. Approximately
300 fish were used in
the investigations during four summers.
RESULTS
Adaptation
illuminated
to constantly
backgrounds
Rates of adaptation observed were in
general in agreement with those reported
by Sumner ( 1911) and Osborn ( 1939).
Average time for adaptation to white background was 24 to 36 hr with a range of 12
to 76 hr. Adaptation to black background
required less time, showing an average of
12 hr with a range of one to 24 hr. Noticeable adaptive changes could be seen within
5 min, but the above intervals were needed
for complete adaptation.
Characteristic
of the black background
adapting sand flounder is the presence of
distinctly circular spots, made up of more
densely packed mclanophores, distributed
over the surface of the animal including
the fins. These spots can be seen as indistinct remnants in the pale fish, As dark
adaptation proceeds, the spots darken relatively rapidly, followed by gradual general
darkening of the whole integument,
Similarly, during white adaptation, white spots
appear. These are less regular in outline
and were reported by Osborn (1939) to be
due to high concentrations of guanine rcla-
R235
SANDFLOUNDER
tivc to the number of melanophorcs.
Aggregation of melanocyte pigment uncovers
the guanophores.
When white adapted fish are disturbed
by any stimulus, they usually show the
phenomenon of “excitement darkening” in
whic!h a rapid (within seconds) dispersion of
melanocyte, pigment occurs. First to respond are the circular spots of concentrated
melanophores.
Denervation
Three types of operations were performed
on fish adapted to a white background:
1)
cutting of the sympathetic chain at definite
points along its course in the posterior part
of the body; 2) cutting of spinal nerves
with the associated sympathetic
fibers
along their axes in the flank of the animal;
3) cutting the extensions of these nerves
into the fin rays.
1) The sympathetic chain was sectioned
by a single stab with a sharply pointed
scalpel just below the vertebral column.
Success in cutting the chain was noted by
the immediate beginning of darkening due
to melanophore dispersion which fanned
out over the animal distal to the cut, Full
intensity of darkening was apparent within
a minute and persisted, to some degree, for
over a week, followed by considerable fading. Freedom of damage to the spinal cord
by the operation was indicated by normal
motor activity in the trunk and tail associated with swimming movements (Plate
1: 2).
In such an operation, special effort was
made to sever nerves completely without disturbing the circulation unnecessarily. Some
damage to the circulatory system, of course,
tAll
figures
are unretouched
photographs
PLATE 1
of the sand flounder
Scopthalamus
aquoms.
1. Photomicrographs
of melanophorcs.
Magnified
x 200.
2. White adapted fish photographed
5 min after sectioning of sympathetic chain.
3. White adapted fish photographed
15 min after sectioning of spinal nerves.
4, 5. White adapted fish photographed
30 min after sectioning of fin rays.
6. White adapted fish photographed
5 min after application
of pressure block for 1 min to lateral
aspect of tail.
7. White adapted fish photographed
15 min after application
for 1 min of cold pack to part of fin
and tail.
8. Same fish photographed
2 hr later.
13236
GEORGE
T. SCOTT
COLOR
CHANGE
IN
TIIE
is inevitable because of the intimate association of the sympathetic chain and the
caudal blood vcsscls in the hcmal canal. The
amount of bleeding in many experiments
was, however, small. The extent of hcmorrhagc appeared to have no influence on the
resulting degree of melanocyte dispersion.
2) Sectioning of spinal nerves resulted in
a dark band distal to the cut extending
across the surface of the fish into the pcriphery of the fin (Plate 1 : 3). Given
sufficient time (several days) the intensity
of the dark area was greatly reduced, indicating lessened chromatophore dispersion.
3) Cutting a fin ray similarly resulted in
a dark band distal to the cut (Plate I : 4,
5). These bands faded out after one to four
days. Although the circulation in the region of the band was interrupted to some
degree by this kind of experiment, ncvertheless, some collateral circulation was observed microscopically.
Continuity of some
circulation in thcsc experiments was further
substantiated by the observation that following systemic injection of epincphrine,
dark bands produced by nerve sectioning
rapidly faded.
Considerable attention was dircctcd to
fin ray recutting distal to the first cut in a
band that had faded (Parker’s experiment).
Generally, if the second and distal cut was
narrow and did not extend laterally beyond
the first cut, a dark secondary band failed
to occur. Occasionally some degree of darkening was noted distal to the second cut.
Although there was variation in the results
obscrvcd in these experiments, equally apparent was the lack of a distinct secondary
dark band following
a second cut made
distal to an carlicr one in a previously faded
SAND
FLOUNDER
R237
band. Unlike the observation reported by
several investigators working on other tclcost fishes, Scopthalamus aquosz~ does not
exhibit a typical positive Parker effect.
Nerve blocking
Spinal nerves, in fish adapted to a white
background, were blocked by pressure and
also by chilling.
A pressure block was imposed by clamping a hemostat to the lateral aspect of the
tail at the junction of the fin for a period of
approximately 1 min. Two layers of adhesivc tape were wrapped around each jaw of
the hemostat to prevent pcrmancnt injury
and the instrument was closed at the first
lock position, During the experiment, the
fish was manually restricted in its movcment to prcvcnt dislodging the hemostat.
Dark banding began immediately distal
to the point of application of the hemostat
(Plate 1 : 6). Within an hour, the bands
began to fade and were completely gone
after several hours. Cutting a fin ray in the
faded arca resulted in a typical dark band
and resembled that caused by a pressure
block.
A cold block was imposed by chilling a
small area of the fins at their junction with
the body. To effect the block, a wedgeshaped piece of solid COB wrapped in two
or three layers of cloth was gently held
against the surface of the fish for approximately ‘l/z min. Localized chilling was facilitated by maintaining the portion of the
fish to which the solid COZ was applied
flush with the surface of the seawater,
Darkening occurred distal to the block in
Icss than 5 min. In three of the six fish used
for this cxpcrimcnt, fading of the bands
PLATE
1
9, 10, and 11. Elcctrodcs placed on cut end of sympathetic
chain, electrical
stimulus applied for 15
set and photographed,
and photographed
1 min after stopping stimulation.
12. Dark band resulting from the injection of 100 pg fluphenazinc
in spinal cord.
13. Black adapted fish, photographed
immcdiatcly
after injection of 100 pg acetylcholine
in spinal
cord.
14. Black adapted fish, 20 ,ug eserine injected in spinal cord.
15’. Black adapted fish, 0.05 pg epincphrine
injcctcd subcutaneously
in opcrcular area (arrow).
16. White adapted fish, 0.5 lug fluphcnazine
injected subcutaneously
(arrow).
R238
GEORGE
occurred within 2 hr; the others required
somewhat longer (Plate 1 : 7, 8).
Nerve stimulation
Pin electrodes were constructed by soldering small sewing needles to fine flexible
wire and then embedding the electrodes
with epoxy resin in drawn-out glass tubing
so that the tips were about 1 mm apart. The
electrodes were then inserted into an incision made to section the sympathetic chain
and positioned in close proximity to the cut
end of the chain (Plate 1 : 9). Tetanic stimulation at 200 v was delivered from a student
electronic stimulator (Harvard Apparatus
Company). Blanching of the darkened area,
which had resulted from sympathetic chain
sectioning, began immediately and persisted
as long as the stimulation was maintained
(Plate 1 : 10). On stopping the stimuIation,
darkening occurred distal to the incision
within a few seconds (Plate 1 : 11).
To further explore the influence of electrical stimulation of nerves containing pigment aggregating fibers, the following three
experiments
were performed
on white
adapted fish:
A cut, slightly less than 0.5 inch (1.27
cm) long, was made in the fish about 1.5
inch (3.8 cm) from the fin margin, resulting in the characteristic dark band extending to the periphery of the fin. First, an
indifferent electrode (a small copper plate)
was placed bcncath the head end of the
fish, a needle electrode in the brain, and
tetanic stimulation applied. No blanching
was observed in the denervated area and
the margins of the band remained clear and
constantly dark. If a black adapted fish
was stimulated in such a manner, the whole
animal lightened during the application of
the stimulus. The electrode was then placed
just distal to the cut on the dark band,
and stimulation resulted in the blanching
of the entire band area for as long as stimulation was continued. The final experiment
consisted of preparing a fish with a dark
fin-ray band by cutting the ray at the central margin of the fin. Stimulation of the
brain, as in the first experiment, did not
result in blanching of the band, whereas
T.
SCOTT
direct application of the needle electrode to
a fin ray resulted in immediate lightening
of the band. Redarkening occurred immediately after stopping the stimulation.
PHARMACOLOGY
The influence of drugs on chromatophore
activity was investigated centrally by injection in the spinal cord and peripherally
by subcutaneous injection.
Injection
in spinal cord
A variety of unrelated drugs, including
the phenothiazine
ataraxics, chloropromazine and fluphenazine; the energizers, imipramine and pheniprazine;
meprobamatc;
prostigmine; atropine; adrenalin and isopropylarterenol, were injected in the spinal cord
in 0.1 ml of fish Ringer. The amount of drug
injected ranged from 100 to 500 pg and the
approximate minimum effective dose ( ED )
was determined. All the above drugs caused
a marked temporary dark band to spread
across the flank of the animal and into the
fins, lasting from less than 5 to as much as
45 min (Plate 1 : 12). Occasionally, all of
the fish posterior to the band darkened to
some degree. The area of melanocyte dispersion seemed to correspond to spinal
nerve distribution.
Exceptional drugs were
acetylcholine, eserine, and serotonin, which
were without effect.
When fish adapted to a black background were used, acetylcholine and eserine
produced a unique result. These were the
only drugs found which would cause light
band formation indicating pigment aggregation. Injection of 100 pg of acetylcholine or
20 pg of eserine resulted immediately in a
transverse light area lasting approximately
1 cand 10 min, respectively
(Plate 1 : 13,
14). Interruption
of the circulation system
by sectioning of the aorta did not prevent
these reactions.
Subcutaneous
injection
The local action of drugs on chromatophores was investigated by subcutaneous
injection in the opercular region which was
found to be the most favorable area for
COLOR
OHH
CHANGE
IN
THE
R239
FLOUNDER
Serial dilutions of the drugs were made
in fish Ringer immediately before use
and injected (0.1 ml volume) subcutaneously
in different parts of the opercular area in a
single fish. The lowest concentration resulting in a positive reaction was then diluted
by tenths and injected in another fish to determine the lowest effective dose. The solution containing this concentration was then
injected into five fish. If all five were positive, the next lowest dilution was tried.
The minimum effective dose (ED ) was defined as that giving a positive reaction in
three or four out of five fish. Some of the
light and dark spots thus produced by this
assay method lasted for several hours, the
persistence depending in large part on the
dosage. The most transient spots recorded
faded in 15 min. Evaluation of drug action
in this manner gave highly reliable data
with little variation among different fish.
Repetition of the determinations one month
later and during the following summer on
entirely different batches of fish gave identical findings.
OH H H
H
SAND
LIP
METANEPHRINE
OCH3
I
MAO
MAO
J
OH 0
o
H
OH 0
DIHYDRO;$,$ANDELIC
MAO
HYDROXYACID
MAO
T
f
OH H
o
Ii
3-METHOXY-4
MANDELIC
OCH3
OH H
NOREPINEPHRINE
OCH3
NORMETANEPHRINE
FIG. 1. First steps in the metabolism
nephrine and norepinephrine.
of epi-
critical observation.
The high degree of
sensitivity demonstrated in this region is
undoubtedly due to a fortunate anatomical
situation.
The opercular area is isolated
from posterior regions by the gill cleft and
from anterior regions by a fold of tissue,
while the immediately underlying opercular
bone further prevents diffusion of the drug
away from the site of injection. A positive
reaction was defined as a distinct color
change (lightening or darkening) of part of
the opercular region within a period of 5
min.
H
H
H
Drugs causing localized pigment
aggregations
A total of 12 drugs, among the many
employed in the investigation, resulted in
pigment aggregation as evidenced by light
spot formation when injected subcutane-
H
OH H
d-&COO”
I:
i
Ii,,
DOPA
:
DOPAMINE
5-HYDROXYTRYPTOPHAN
FIG. 2.
Metabolic
precursors
H” NOREPINEPHRINE
5-HYDROXYTRYPTAMINE
(SEROTONIN)
of norepinephrine
and Shydroxytryptamine
( serotonin)
.
1~240
GEORGE
T. SCOTT
TABLE 1. Drugs causing local melanophore
pigment aggregation when iniected subcutaneously
in the
opercu.lar area, and the influence of pretreatment
with pyrogallol.
Some inactive analogs are included.
Figures represent minimum effective dose for a positive reaction in three or four out of five fish
---. -.-___
AmouT;zectecl
Amount injected (fig)
after pyrogallol
L-Epincphrine
D-Epinephrine
L-Norepincphrine
DL-Norepinephrine
Isopropylnoradrenaline
Dichloroisopropylnoraclrenaline
DL-Metancphrine
L-Mandclic
acid
0.04
0.04
0.08
0.10
0.08
7.0
inactive
inactive
0.00010
0.00008
0.00006
0.00002
7.0
-
Dopamine
Dihydroxyphenylalanine
5-IIydroxytryptaminc
5-IIydroxytryptophanc
0.02
inactive
0.04
inactive
0.02
2.0
-
Methamphctamine
0.04
0.04
Phenclzinc ( Nardjl)
Pheniprazine
( Catron )
E tryptamine
( Monase )
Iproniazid
(Marsilid)
Isocarboxazid
(Marplan)
0.3
0.5
2.0
inactive
inactive
0.4
-
( dopa)
(serotonin)
ously in the opercular region of black
adapted fish (Plate 1 : 15). Analogs of some
of the drugs were included.
The most potent pharmacological agents
were epinephrine,
norepinephrine,
dopamine, serotonin, and methamphetamine
(ED 0.02 to 0.08 pug). The analogs of norepinephrine, isopropylnoradrcnalinc,
and dichloroisopropylnoradrenaline
were least active. Complete inactivity was shown by the
normal metabolic derivatives of epinephrinc, metanephrine, and mandelic acid (Fig.
1). Similar inactivity
was shown by the
metabolic precursors of dopaminc and serotonin, dihydroxyphenylalanine
(dopa), and
5-hydroxytryptophane
(Fig. 2). Three of the
five drugs in the monoamine oxidase inhibitor category, phcnelzine, pheniprazine, and
etryptamine were active at higher levels
(ED 0.3 to 2 pg). Iproniazid and isocarboxazid were inactive ( Table 1) .
Drugs causing localized pigment dispersion
Approximately
30 drugs were found to
produce a dark patch in the opercular skin
of light fish. Fourteen drugs, representing
four different categories, are included in
this report. Eight are members of the phenothiazine tranquilizer family (fluphenazine,
perphenazine, thiopropazate, proclorpromazine, chlorpromazine, trifluoperazine, acetophenazine, mephazine); two are mild tranquilizers (chlordiazepoxide,
meprobamate);
two are adrenergic blocking agents (dibenamine, ergotamine tartrate), and one a local
anaesthetic ( dibucaine) .
By far the most active melanin dispersing
drugs were certain of the phenothiazines
(ED 0.08 to 0.10 pg), whereas the least
potent of all drugs possessing pigment dispersing activity were the mild tranquilizers
followed by the barbiturates (ED 80 to 100
pg) (Table 2).
Acetylcholine
and escrine gave negative
results, up to the limit of dose injected ( 100
pg ), in both light and dark fish.
Influence of p yrogallol
The principal
enzyme in some tissues
catalyzing the physiological inactivation of
catechol amincs, such as epinephrine and
norepinephrine, has been established to be
catechol-o-methyl
transferase
(COMT).
Pyrogallol is an inhibitor of this enzyme. It
COLOR
CHANGE
IN
pigTAULE 2. Drugs causing local melunophore
ment dispersion when injected subcutaneously
in
the opercular area, and the in;fluence of pretreatment with pyrogallol.
Figures represent minimum
effective
dose for a positive reaction in three or
four out of five fish
Fluphenazine
( Permitil )
Perphenazine
( Trilafon )
Thiopropazate
( Dartal )
Dibenamine
Ergotamine
tartrate
+nount
Amount
injcctcd
(/.a)
(g$%&
pyrogallol
0.08
0.09
0.1
0.2
0.3
7
4
10
9
10
Dibucaine
0.2
Prochlorperazinc
( Compazine )
0.7
Chlorpromazine
(Thorazine)
0.8
Trifluoperazine
( Stelazine)
4
hcetophenazine
( Tindal)
Mepazine
( Pacatal )
5”
Chlordiazepoxide
( Librium )
70
Mcprobamate
(Miltown)
80
Phenobarbital
sodium
100
0.2
0.G
0.8
4
5
5
70
80
100
was therefore gcrrnane to consideration of
the possible transmitter action of catechol
amincs at the chromate-neural junction to
explore the influence of the enzyme inhibitor on the ED of injected drugs required
for pigment aggregation or dispersion. Accordingly,
white and black background
adapted fish were pretreated by intrapcritoneal injections of pyrogallol (5 mg/kg)
0.5 hr before drug evaluation. The systemic
injection of pyrogallol had no influence per
se on the coloration of a fish but resulted
in a drastic reduction in the ED of epinephrine and norepinephrine for pigment aggrcgation. The value for scrotonin, on the
other hand, was significantly elevated while
that of the other two drugs tested, methamphetamine
and phenelzine,
was unchanged (Table 1). Complementary expcriments were carried out on white background
adapted fish. The ED of five of the pigment dispersing drugs was raised by 1 to 2
magnitudes and the others remained unchanged ( Table 2).
Inf hence of pituitary hormones
Injection of commercial pituitrin
(Parke
Davis and Company) subcutaneously in the
opcrcular region of light fish caused no pig-
TIIE
SAND
R241
FLOUNDER
ment dispersion. The melanophores were
likewise found to be refractory to pure hog
p-MSII up to the maximum quantity injected (100 pg, 5 X lo5 Shizume-Lerner
units).
DISCUSSION
The cxpcriments of Osborn (1939) on the
sand flounder, in which the sympathetic
chain was scvcrcd or nerve cuts were made
in the flank, together with electrical stimulation of the central nervous system, strongly
suggested the presence of pigment aggregating nerve fibers. The problem remains,
however, as to whether dispersing nerve
fibers were functional in the sand flounder.
Osborn was inclined to doubt their presence.
The neurological and pharmacological cxperiments described in this report were designed to extend the range of observations
relating to this mechanism of chromatophorc
control. Particular attention was given to
the question of mono- or dincuronic innervation as a background for consideration of
drug action on melanocytc activity.
The marked and prompt melanocyte dispersion following
interruption
of sympathetic nerve supply brought about by sectioning of the sympathetic chain or spinal
nerves and the prompt aggregation of pigmcnt following direct electrical stimulation
of the cut nerves, confilms the prescncc of
active pigment aggregating nerve fibers.
The blanched state characteristic
of the
light-background
adapted fish is obviously
maintained by the tonic discharge of these
nerve fibers. Imposing a cold or pressure
block results in the expected pigment dispersion distal to the block because of lessened
activity of the fibers. The presence of aggregating nerve fibers therefore is beyond
doubt.
Considerable effort was made to find
evidence indicating the activity of the dispcrsing fibers which have been claimed to
be operative in several other teleosts. In
these fish, the strongest suggestion of pigment dispersing fibers was derived from the
cxperimcnt in which recutting distal to a
previous cut in a faded band resulted in
secondary darkening, due presumably to rc-
R242
GEORGE
stimulation by injury of dispersing fibers
(Parker effect). Such a second cut in the
fin or flank of the sand flounder, if the cut
dots not extend laterally beyond the earlier
and more central cut, does not typically result in secondary darkening, The occasional
exceptional response in which some partial
secondary darkening occurs could possibly
have been caused by cutting one or more
overlapping nerve fibers not severed by the
first cut. Exploration
of this possibility
could be followed by careful histological
procedures designed to reveal the pattern
of innervation,
especially in the interray
membrane.
Circulatory congestion caused by incisions
made in severing nerves could also, to some
degree, be a contributory factor to melanocyte pigment dispersion. It is conceivable
that the background level of pigment aggregating substances present in the blood
would be reduced in the area of partial circulatory arrest. The added congestion, due
to a second cut in a band in which circulation had been partially reestablished, could
be a factor causing some secondary dispersion of pigment. The presence of aggregating substances in the blood may explain
the observation that dark bands resulting
from cutting a single fin ray fade much
sooner ( one day or less) than bands resulting from longer and more drastic cuts that
may require several days for fading on a
light background. That some non-nervous
factor may contribute to melanocyte dispersion distal to a second cut was suggested by
Osborn (1939), who observed some secondary darkening in flatfish as long as four
weeks after an earlier and more central cut
had been made; reactivation of dispersing
nerve fibers was unlikely, because in all
probability
only degenerated nerves were
present in the area involved.
Incidentally,
the time for fading of dark
fin-ray bands in Scopthalamus aquosus is
much less (one to four days) than in Fundulus and other forms ( several days) on
which dispersing fibers have been postulated. Fading of the bands could result
from a greater sensitivity, as a result of de-
T.
SCOTT
nervation, to circulating pigment concentrating agents in the blood.
A striking difference between Fund&us,
in which dispersing nerve fibers have been
postulated, and the sand flounder is the response to localized chilling.
Parker (1936)
reported that dark band formation, following nerve cutting, would not extend through
and beyond an area of the fin subjected to
a cold block. This was interpreted as due
to a conduction block imposed on dispersing
nerve fibers. The sand flounder shows a
quite different response. Dispersion of pigment always occurs distal to a cold (or pressure) block, presumably due to lessened
activity of aggregating fibers. A similar
cold block applied to a black-background
adapted fish has no effect; at least no pigment aggregation occurs such as might follow blocking of dispersing fibers if they
were present.
The variety of unrelated drugs injected
into the spinal cord of light-background
adapted fish apparently had an inhibitory
influence on neurological activity in the central nervous system resulting in a lessened
discharge over the pigment concentrating
fibers and therefore in pigment dispersion
(Plate 1 : 12). Acetylcholine,
eserine, and
serotonin appeared not to have such an inhibitory effect because the dark band did
not appear.
When black-background
adapted fish
were used, the uniqueness of acetylcholine
and eserine was apparent. These drugs
probably brought about cholinergic facilitation in the cord, causing greater discharge
over the sympathetic nerve fibers affected
and resulting in the light band across the
flank of the fish ( Plate 1 : 13, 14).
Examination of the structure of the compounds found to be highly active as melanophore pigment aggregators, when injected
subcutaneously in the opercular area, reveals a common chemical homology. Almost
all are substituted aromatic ethyl amines.
The two exceptions are aromatic ethyl hydrazines.
A high degree of group specificity is involved, indicated
by the inactivity
of
closely related compounds. Metanephrine
COLOR
CHANGE
IN
and mandelic acid represent the first steps
in the normal metabolism of epinephine and
norepinephrine (Fig. 1). Both of these compounds were found to be inactive. A similar refractoriness was shown toward dopa
and $hydroxytryptophane,
the immediate
metabolic precursors of dopamine and serotonin, respectively (Fig. 2). The two active
drugs in the monoamine oxidase inhibitor
category, phenelzine and pheniprazine, are
the two without substitutions on the hydrazinc group. Substitutions on this group as
in the case of iproniazid and isocarboxazid
apparently rendered these compounds inactive (Table 1).
The high activity of epinephrine was to
be expected in view of its general pigment
aggregating property. Relatively little was
known about the action of norepinephrine,
although a strong pigment concentrating effect was reported by Umrath (1957) in the
bitterling and by Fujii ( 1961) in the goby
Chasmichthys.
The high activity of dopamint could result from its similarity to norepinephrine or its conversion to this compound by the skin tissue. Methamphetamine
is a potent sympathomemetic amine simulating the action of epinephrine in many
physiological reactions. The action of the
two hydrazines, phenelzine and pheniprazine, probably is not due to amine oxidase
inhibition
because iproniazid
and isocarboxazid are also MAO inhibitors and are
inactive. These latter two drugs are, however, less potent MAO inhibitors than are
phenelzine and pheniprazine (Randall and
Bagdon 1959 ) .
The action of pyrogallol on melanophore
physiology was especially interesting and
significant because of the observed drastic
potcntiation of cpinephrine and norepinephrinc (Table 1). Pyrogallol inhibits the methylation of catechol amines by acting as a
competitive substrate for catechol-o-methyl
transferase ( Axclrod and Tomchick 1959;
Undenfriend et al. 1959) and has been shown
to inhibit this enzyme in fish (Scheline 1962).
The inhibitor has been observed to potcntiate the action of exogenous epinephrine
(Bacq
et al. 1959; Wylie, Archer, and Arnold
1960) and to markedly inhibit the disappear-
THE
SAND
FLOUNDER
R243
ante of this amine in mice (Axelrod and
Laroche 1959). The marked reduction in
the ED of injected epinephrine and norcpinephrine suggests the active participation of
the enzyme in the metabolism of catechol
amines in the skin of Scopthalamus aquosus.
The presence of catechol-o-methyl transferase would strongly strengthen the probability of a catechol amine functioning
as
the normal transmitter at the chromatoneural junction.
The high potency of serotonin as a pigment aggregator is in contrast to the action
of this compound in the goby Chasmichthys
for which Fujii (1961) reported that neither
normal or denervated melanophores responded with pigment concentration.
The
remarkable increase in the ED of 5-hydroxytryptamine in the sand flounder following
pretreatment with pyrogallol suggested a
different mechanism for the action of the
epinephrines and serotonin. Additional research on this reaction is indicated.
The drugs producing localized pigment
dispersion when injected subcutaneously
arc numerous and diverse in structure.
Those included in this report are pharmacalogically in the tranquilizer, depressant, and
sympatholytic categories. The diversity in
structure suggests that many have some
general influence resulting in the “relaxation” of the melanophore to the “expand&i”
state. However, certain of these drugs
whose action is elucidated by pretreatment
with pyrogallol appear to act in a more
specific manner.
The three most active pigment dispersing
drugs are members of the phenothiazine
tranquilizer group (fluphenazine, perphenazine, thiopropazate ) . These drugs together
with ergotamine and dibenamine are the
ones whose ED is markedly raised after the
fish have been previously injected with pyrogallol (Table 2).
Following
the classic investigations
of
Dale, ergot alkaloids have been used extcnsively as adrenalin blocking agents. Included in the numerous studies of adrenalin
reversal by these compounds has been the
melanophore system of a number of fishes
(Spaeth and Barbour 1917; Smith 1931; Veil
11244
GEORGE
T.
SCOTT
ings and physiological
jnactivation of the
1936; Parker 1941a). Fujii (1961) observed
the reversal of the pigment aggregating ef- transmitter by catechol-o-methyl transferase.
fect of adrenalin on both innervated and
SUMMARY
AND
CONCLUSIONS
denervated melanophorcs of the goby Chasmichthys and concluded that crgotamine
1. The mechanisms of chromatophore conacts directly on the effector cell without introl in fishes are discussed,
tcrcalation of neural mechanisms.
2. Evidence for pigment concentrating
Dibenamine is one of the p-haloalkylanerve fibers in the sand flounder, based
mines found to be a potent adrencrgic blockon nerve cutting, nerve blocking and
ing agent, the mode of action of which
nerve stimulating experiments, is prehas been carefully analyzed (Nickerson and
sented.
Goodman 1947; Nickerson and Nomaguchi
3. The presence of functional
pigment
1948). From these studies the conclusion is
dispersing nerve fibers is assumed unreached that, in the process of the establishlikely because of the absence of the
ment of excitation to adrenergic stimuli, ditypical “Parker effect” following nerve
benamine blocks receptor sites which are
recutting.
different than those required for the action
4. A number of unrelated drugs, when
of other directly
stimulatory
substances
injected into the spinal cord of light
(Nickerson and Nomaguchi 1948).
fish, cause pigment dispersion followThe fact that three of the phenothiazine
ing spinal nerve distribution.
Acetyltranquilizers act similarly to the adrencrgic
choline, eserine, and serotonin do not
blockers, in terms of potency and change in
have this effect.
ED following pretreatment with pyrogallol,
and cserine were the
5. Acetylcholine
suggests a common mechanism. Presumably
only drugs, which when injected into
these drugs interfere with the interaction of
the spinal cord of dark fish, caused
the normal catechol amine transmitter on
pigment aggregation following spinal
the melanophore required for the maintenerve distribution, presumably because
nance of pigment aggregation. The inhibiof enhanced excitation of concentrating
tor action of pyrogallol on catechol-o-methyl
nerve fibers. The reaction occurred
transferase would be such as to elevate the
even under conditions of complete cirlevel of endogenous catechol amines. The
culatory arrest.
amount of injected blocking agent required
6. The opercular region was found to be
for competitive reaction with receptor sites
a highly reliable area for the study of
to produce marked pigment dispersion
drugs causing localized melanophore
would thereby be raised. Such an interprepigment aggregation or dispersion.
tation would explain why the above three
7. All drugs causing pigment aggregation
tranquilizer drugs have the highest potency
when injected subcutaneously
were
of all drugs tested as pigment dispersing
substituted aromatic ethyl amines or
agents. Such a possibility reinforces the hyhydrazines.
The most potent were
pothesis that a catechol amine (epincphepinephrine,
norepinephrine,
doparine, norepinephrine,
or dopamine) is the
mine, and methamphetamine.
neurohumoral
agent involved in pigment
8. A large number of drugs caused pigaggregation.
ment dispersion when injected subOne would assume, therefore, that the
cutaneously. The most potent were the
phenothiazinc tranquilizers
(fluphenaextent of pigment concentration or dispcrzine, perphenazine, thiopropazate) .
sion in the melanophores of Scopthalamus
9. Pretreatment of the fish with pyrogallol,
aquosus would depend on the amount of enan inhibitor of catechol-o-methyl transdogenous catechol amine transmitter availferase,
drastically potentiated epinephable to active sites on the melanocytc.
rine and norepincphrine.
This would depend on the balance between
neurosccretion bv concentrating nerve end- 10, Pretreatment with pyrogallol increased
COLOR
CIIANGl3
IN
the minimum effective dose of certain
pigment dispersing drugs. Three of the
drugs so affected were phenothiazinc
tranquilizers
and two were adrenalin
reversing drugs. The cffcctive dose of
the other pigment dispersing drugs was
unchanged following pyrogallol.
Il. The assumption is that all five pigment
dispersing drugs, whose effective dose
was raised by pretreatment with pyrogallol, cause pigment dispersion by
adrcnergic blockage.
12. The pharmacological
observations involving the local action of drugs strongly
suggest that a catechol amine (epinephrine, norepinephrine,
or dopamine) is
the transmitter at the chromate-neural
junction and that catechol - o -methyl
transfcrasc is involved in chromatophore
physiology.
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