AM. Zooi.rx.isT, 9:489-501 (1969).
Physiological Color Changes in Reptiles
MAC E. HADLEY AND JOEL M. GOLDMAN
Department of Biological Sciences, and Division of Pharmacology,
University of Arizona, Tucson, Arizona 85721
SYNOPSIS. The physiological regulation of color changes in reptiles as studied in the
lizard, Anolis carolinensis, is discussed. In Anolis, the ability to adapt to a background
is dependent upon the level of circulating MSH, the release of which is dependent on
information received through the eyes. Blinded (or intact) lizards are brown under
conditions of strong illumination and green under conditions of lower light intensities,
and, again, these color changes are regulated by MSH. According to Kleinholz, color
changes in the blinded lizard are regulated by dermal photoreceptors. High or low
temperatures directly affect the color of Anolis skins and alter the rate at which skins
respond to hormones. Aggregation of melanin granules within Anolis melanophores in
response to sympathomimetic stimulation is regulated through alpha adrenergic receptors whereas dispersion of melanin granules in response to such stimulation is
controlled through beta adrenergic receptors possessed by the melanophores. Most
Anolis melanophores possess both alpha and beta adrenergic receptors, but some
melanophores possess only beta adrenergic receptors. In the normal physiology of the
lizard, under conditions of stress, stimulation of alpha adrenergic receptors by
catecholamines leads to an "excitement-pallor" followed by an "excitement-darkening"
resulting from stimulation of beta adrenergic receptors which causes dispersion of
melanin granules within localized populations of melanophores. Thus, in Anolis,
dispersion of melanin granules within melanophores is regulated by both MSH and by
catecholamines. Evidence is presented that the intracellular level of cyclic 3', 5'-AMP
within melanophores may be responsible for the regulation of movement of melanin
granules.
Since the studies of Kleinholz (1936,
1938a, b) on the lizard, Anolis carolinensis, there have been few investigations of
the nature of chromatophoric regulation
in reptiles. Therefore, most references on
the subject are already provided in the
earlier monographs by Parker (1948), Fingerman (1963), and Waring (1963).
Most studies on mechanisms of color
change in reptiles have been confined to
the African chameleons, Chamaeleo pumilus and C. vulgaris; the horned lizards,
Phrynosoma blainvilli and P. cornutum;
and the Florida anolid, Anolis carolinensis. Although the chromatophoric responses of these reptiles are similar in a number of respects, enough differences exist
This study was supported in part by USPHS
Research Grant No. 2-F2-AM-32, 622-02 from the
National Institute of Arthritis and Metabolic Diseases, GB-3681 from the National Science Foundation, and a National Institutes of Health Institutional Grant to the University of Arizona.
between the species to make generalizations difficult.
Briicke (1852) early demonstrated that
color changes in the chameleon, C. pumilus, were under sympathetic nervous regulation. According to Hogben and Mirvish
(1928) there is no evidence for hormonal
regulation of chromatophores in chameleons. In Phrynosoma, both nerves and hormonal agents (Redfield, 1916, 1918; Parker, 1938) appear to play a role in regulation of chromatophores. In Anolis carolinensis, in contrast, the careful studies of
Kleinholz (1936, 1938a, b) demonstrated
that only hormonal agents are involved in
chromatic control in this lizard.
Unfortunately, a clear interpretation of
the literature on regulation of chromatophores in reptiles, as in other vertebrate
groups, is often confused by the rather
dogmatic opinions expressed by a number
of the early leading investigators in the
field.
489
490
MAC E. HADLEY AND JOEL M. GOLDMAN
The present communication discusses
the nature of chromatic regulation in the
lizard, Anolis carolinensis, the only reptile
on which there has been continuing research. Fortunately, the pioneering work of
L. H. Kleinholz provided a wealth of data
on the mechanisms of chromatophoric regulation in Anolis carolinensis, which, along
with more recent data (Horowitz, 1957,
1958; Goldman and Hadley, 1969) provide
a rather clear basis for an understanding
of chromatic regulation in this reptilian
species. Jn fact, there are few other vertebrate species for which such complete
data on mechanisms of color change are
now available.
HISTORICAL SUMMARY OF COLOR CHANGE
RESPONSES OF Anolis carolinensis
Unless otherwise cited, the following
data have been summarized from the
studies of Kleinholz (1936, 1938«, b). Anolis carolinensis adapts readily to a light- or
to a dark-colored background, being
bright green on the former and a dark
brown on the latter. The movement of
melanin granules within the dendritic processes of the dermal melanophores is largely, if not entirely responsible for color
change (von Geldern, 1921). The brightcolored pigment cells, the iridophores and
xanthophores, overlying the melanophores
probably play a passive role in color
change. Reflectance of the shorter waves of
light from the reflecting platelets within
the iridophores and their passage through
the overlying yellow layer of pigment cells,
the xanthophores, results in a green color
being seen. The green color of the lizard
can only be manifested when light can
reach and be reflected from the iridophores lying above the melanophores. The
dispersion of melanin granules into the
dendritic processes of the melanophores
which terminate above the iridophores
prevents such penetration, resulting in the
skins becoming dark brown.
Although Anolis will adapt readily to a
background, the intensity of overhead
lighting is apparently an important factor
in the regulation of color change. On a
neutral background with strong overhead
lighting, lizards are usually dark brown.
They are usually bright green under general laboratory conditions of fluorescent
lighting. If taken out into strong sunlight
they rapidly become dark brown. If lizards that are dark brown are transferred
to darkness, they become bright green
(Carlton, 1903; Parker and Starratt, 1904;
von Geldern, 1921). Blinded lizards lose
their ability to adapt to a background, but
can still respond to the presence or absence
of light. Blinded lizards are dark brown
under conditions of overhead lighting,
whether on a light- or a dark-colored background. They become bright green if
transferred to the dark. Kleinholz suggested that either the melanophores are
directly responsive to light or that some
photoreceptor other than the eyes is involved. Hypophysectoinized lizards lose
their ability to adapt to a background and
are bright green under all conditions of
illumination. Thus, the melanophores of
the intact skin do not appear to be directly
sensitive to light. However, Hadley (1928,
1931) demonstrated that isolated skins
from Anolis eqnestris, A. iodurus, A. watsoni, and A. carolinensis show direct responses to light. Skins are of varying degrees of brown in direct sunlight but under
diffuse illumination are green. Smith
(1929) obtained similar results on isolated
skin of A. eqnestris and Kleinholz
confirmed the results of Hadley (1931) on
A. carolinensis. The response of A. carolinensis skins to strong light is slight, however. Because a similar result was not obtained with hypophysectoinized lizards, it
was concluded that direct responses of melanophores to light probably do not account for the color response of lizards to
the presence or absence of light.
Garlton (1903) showed that light which
fell on one part of the lizard's body results
in darkening in the other non-illuminated
portions of the body, indicating an indirect
effect of light on the melanophores. These
results also revealed that a direct stimulation of the parietal eye, pineal organ, or
COLOR CHANGICS IN RKI'TILKS
associated area of the epithalamus by the
presence or absence of light does not regulate chromatic behavior. Lizards remain
brown if the head is covered but the body
exposed to illumination, or if the body is
covered and only the head illuminated. II
the circulation is excluded from any part
of Anolis, the skin in that area becomes
green, as does isolated skin placed in vitro
in Ringer's solution. Kleinholz concluded
that the color changes of a blinded anolid
must be due to the stimulation of dermal
receptors which, then, by a spinal reflex,
regulate the release of a hypophyseal chroma tophorotropic factor.
Intermediate lobes from either Rana
pipiens or Anolis itself when injected into
hypophysectomized or normal lizards result
in a rapid darkening of the skins, indicating that a hypophyseal hormone is involved in chromatic regulation. This darkening is completed in only a few minutes,
and the time is similar to that normally
required for a lizard to change from green
to brown. Also, isolated pieces of skin
darkened when placed in solutions of pituitary extract. More recently, Horowitz
(1957, 1958) and Novales (1959) have
shown that preparations of melanophorestimulating hormone, in vitro, cause a similar rapid and maximal darkening of the
skin oi.' Anolis. Novales (1959) also showed
that the response to MSH is markedly inhibited in sodium-free potassium Ringer's
solution, indicating that there is a sodium
requirement for MSH-action on Anolis
melanophores. Novales, Novales, Zinner,
and Stoner (1962) found that this inhibition is correlated with a 90% reduction in
the sodium content of the skins.
Although cutting and destroying the
posterior spinal cord have no effect on the
ability of a lizard to adapt to background,
Carlton (1903) still considered that color
changes were regulated by nerves. May
(1924) also believed, from his transplantation-studies, that color changes in Anolis
were controlled by nerves. Kleinholz, however, clearly demonstrated that autotransplanted and homotransplanted skins respond to the nature of the background in
•191
accordance with the surrounding normal
skin. Injections of "Pituitrin" into individuals carrying grafts resulted in darkening
of the transplanted skin. Furthermore, as
demonstrated by Carlton (1903), Kleinholz also showed that lizards with spinal
transection plus pithing of the lower
spinal cord still are able to adapt to a
light-colored or a dark-colored background. These results demonstrate that
nerves are not involved in the regulation
of changes in color in Anolis. Neither von
Geldern (1921) nor May (1924) was able
to demonstrate innervation of integumental melanophores.
Electrical stimulation of Anolis by various methods gives a number of quite distinct results: (1) green, intact lizards become uniformly dark brown, a result
which Kleinholz interpreted as coming
from a direct stimulation of the pituitary
to release a chromatophorotropic hormone;
(2) in other similar lizards the only observable change is a darkening of the postorbital region and scattered groups of
scales over the otherwise green body,
referred to as the mottled condition; (3)
stimulation of intact, dark animals evokes
a generalized pallor in about two minutes
and this is followed by mottling; (4) stimulation of hypophysectomized lizards results in post-orbital darkening and a generalized mottled pattern. The mottling persists in hypophysectomized lizards for at
least 20 minutes after stimulation. Within
an hour it usually disappears and the lizard is again uniformly green. Post-orbital
darkening resulting from electrical stimulation occurs even if such areas are removed and replaced on the individual,
thus demonstrating that such darkening is
not under direct nervous control.
It was noted that this mottling is associated with conditions of emotional excitement. Epinephrine injected into hypophysectomized animals causes mottling,
as do adrenal extracts from Anolis. Hypophysectomized, adrenalectomized lizards
do not mottle in response to electrical stimulation. Hadley (1931) noted that isolated skin from Anolis iodurus floated onto
492
MAC E. HADLEY AND JOEL M. GOLDMAN
anophores in the lizard, Anolis carolinensis, have involved an in vitro method (Novales, 1959; Goldman and Hadley, 1969)
as originally described by Shizume, Lerner,
and Fitzpatrick (1954) and Wright and
Lerner (1960) for the frog, Rana pipiens.
Horowitz (1957, 1958) has described a
somewhat similar method. In virtually all
respects these data complement the in vivo
data provided by Kleinholz (1936, 1938a,
b) and, in addition, demonstrate a clear
role for catecholamines, probably of adrenal-medullary origin, as important humoral
factors in the direct regulation of melanophores under conditions of stress. In addition, evidence is provided that catecholamines directly control both aggregation and
dispersion of melanin granules within
Anolis melanophores, the latter phenomenon being responsible for the mottled
pattern ("excitement-darkening") of the
skin. Aggregation of melanin granules on
the other hand, in direct response to
catecholamines accounts for the "excitement-pallor" exhibited by A. carolinensis.
In addition, evidence is provided (Goldman and Hadley, 1969) that catecholamines mediate these responses through
stimulation of adrenergic receptors as
classically defined by physiologists. Of
importance will be the demonstration
that dispersion of melanin granules as regulated through beta adrenergic receptors
probably involves stimulation of adenyl
cyclase and the intracellular synthesis of
cyclic 3', 5'-adenosine monophosphate.
The isolated skin of Anolis is admirably
suited for such studies in that in any one
experiment a large number of skins (one
from each lizard) can be removed quickly
and their response to hormonal and/or
pharmacological stimulation monitored
rapidly with a high degree of statistical
significance. When skins are removed and
placed in Ringer's solution they begin to
lighten due to a slow perinuclear aggregation of melanin granules within the dermal melanophores. After a suitable preIn vitro STUDIES ON RESPONSES OF
incubation in Ringer's solution to allow
MELANOPHORES TO HORMONES
the skins to equilibrate, they can then be
More recent studies on regulation of mel- tested for their response to various stimuli.
solutions of epinephrine became brown
from maximum dispersion of melanin
granules within the melanophores. Isolated
skin from A. carolinensis became mottled
in epinephrine, suggesting to Kleinholz the
role of the adrenals in regulating this response.
Epinephrine injected into dark-colored
lizards causes blanching, but adrenalectomized lizards can still become green. From a
consideration of the times involved in responses to background it was considered
that the slow response (20-30 min) to a
white background involved the gradual
disappearance of a chromatophorotropic
hormone (MSH) from the circulation.
Kleinholz came to the conclusion that the
rapid pallor produced by electrical stimulation came from a generalized vasoconstriction "thereby resulting in an effective exclusion of the melanophoredispersing hormone from the pigmentary
effectors."
The fact that melanin granules are aggregated within melanophores after hypophysectomy and in pieces of skin floated
on Ringer's solution indicated to Kleinholz that a second ("W" hormone of Hogben, 1936) or other "as yet unidentified"
hormone is not responsible for normal
lightening. Apparently Kleinholz favored
the unihumoral concept of chromatic control and would not concede the possibility
that a second hormone might be involved
in the direct regulation of movement of
melanin granules within melanophores.
He was apparently interested in demonstrating that neither nerves, nor epinephrine of an adrenal-medullary source, directly regulated melanophore activity. He
stated, "these experiments show that the
contraction of the melanophores is neither
dependent upon the direct action of nerves
nor upon adrenal secretion. The experiments do, however, indicate that blanching
is regulated through the circulation."
COLOR CHANGES IN REPTILES
FIG. 1. Demonstration in vitro of the uniformity of
responses of Anolis skins to MSH. One group (•) of
skins was darkened with MSH for 60 min and then
the Ringer's control group ( • ) was subjected
to a similar concentration of MSH. At 120 min, the
skins in each group were rinsed in Ringer's
solution several times. Each point on the graph is
the mean of eight measurements of reflectance.
Vertical lines represent the standard error of the
mean.
As demonstrated by Horowitz (1957,
1958) and Novales (1959), the skins of
Anolis become maximally dark (dark
brown) in response to melanophorestimulating hormone (MSH). Burgers
(1961) has described a bioassay system for
MSH using isolated pieces of Anolis skin.
In our experiments, we have used porcine
/J-MSH and have found that maximal
darkening involves a drop to about 60 or
50% of their original reflectance (Fig. 1).
The darkening response in any one experiment is usually quite similar between any
two groups of skins. When the skins are
rinsed in Ringer's solution the melanin
granules reaggregate within the melanophores and the skins lighten to their original base values. When skins are then again
subjected to a similar concentration of
MSH, they redarken to the same extent as
in the previous stimulation. A near maximal response is obtained with a concentration of 3 X 10~9 g/ml- Lower concentrations of MSH produce proportionately less
darkening, and a minimal response was
recorded at 1 X 10" 11 g/ml. Similar responses are evoked by porcine a-MSH.
The time-response of Anolis skins in vitro to darkening by MSH, and their lightening in its absence, closely parallel the
493
times observed for this lizard in response to
a light-absorbing (dark-colored) or a lightscattering (light-colored) background (Carlton, 1903; Parker and Starratt, 1904; Kleinholz, 1938a). If Anolis skins are near-maximally darkened with MSH, then, the addition of norepinephrine (or epinephrine)
causes a reaggregation of melanin granules
within dermal melanophores leading to a
rapid lightening of the skins (Fig. 2A) as
first demonstrated in vitro by Horowitz
(1958). This reversal is often maximal
(skins relighten to their original basevalue) with higher concentrations (10-410- 6 M). Lower concentrations (10—7-10—s
M) of these catecholamines give measurable but submaximal lightening (Fig. 2B).
The response is rapid, often being maximal
within two minutes after the catecholamine
is added to the skins. The maximal lightening response is not maintained, and the
skins soon redarken to some extent.
The lightening response of Anolis skins
to catecholamines far exceeds that of skins
of the frog, R. pipiens, to similar stimulation (Hadley and Bagnara, 1969). In addition, all MSH-darkened Anolis skins lighten in response to either norepinephrine
or epinephrine, whereas the individual
response of frog skins varies considerably
from animal to animal. Our cytological observations of Anolis skins stimulated by
hormones have failed to reveal that any
chromatophores other than melanophores
(e.g., xanthophores and iridophores) respond. Dispersion of melanin granules occurs in both dermal and epidermal melanophores. Thus, objective measurements are
reliable evidence for the action of hormones on movement of melanin granules
within melanophores. Similar measurements of changes in reflectance from frog
skins, in contrast, include the response of
the reflecting cells, the iridophores, and
complicate interpretations from hormonal
studies unless the reflectance data are correlated with microscopic observations of
the cellular responses involved (Hadley
and Bagnara, 1969).
Melatonin and acetylcholine are consid-
494
MAC E. HADLEY AND JOEL M. GOLDMAN
15
30
45
TIME, minutes
60
15
30
105
45
60
75
TIME, minutes
FIG. 2. Response in vitro of MSH-darkened skins
of Anolis to varying concentrations of norepinephrine. A. In the first experiment, skins were darkened with MSH (3 X 10"0 g/ml) for 30 min
and then norepinephrine was added at a concentration of either lO"4 (X), 10"5 ( • ) , 10"6 (•), or
10"7 M ( • ) to each group of skins. One group
(O) of skins was maintained as an MSH-control.
B. In the second experiment, a single group of
MSH-darkened skins was subjected to increasing
concentrations (10~9-10~7 M) of norepinephrine. The
skins were then rinsed several times in fresh
Ringer's solution to demonstrate their ability to
further lighten. Each point on the graph is the
mean of eight measurements of reflectance.
ered potent lightening agents of R. pipiens
skins darkened by MSH (Lerner, 1959).
We have been unable to demonstrate such
an effect on Anolis skins when the melatonin was added to the MSH. Melatonin
only minimally lightens Anolis skins darkened by MSH (Fig. 3), and in some experiments is without effect. In these experiments, norepinephrine always lightens
(control response) these skins. Acetylcholine has no noticeable effect on Anolis skins.
Anolis becomes dark-brown in response
to a dark-colored background, but becomes
pale (green) when transferred to the
dark. Blinded lizards also pale in the dark;
thus, the eyes do not regulate this response. Parker (1938) noted a similar response with Phrynosoma blainvilli, as have
numerous other investigators working
with chameleons (Sand, 1935), and Parker
demonstrated that the response took place
whether the parietal eye was covered or
not. Carlton (1903) noted that A. carolinensis is brown if the head is placed within
a black box but the body is still subjected
to illumination. The pituitary is necessary
in Anolis for color change from darkness
to conditions of illumination (Kleinholz,
1938«). The time for paling in darkness
NE(IO"=M)
15
30
45
TIME, minutes
60
75
FIG. 3. Comparison of responses in vitro of melanophores of Anolis to melatonin and norepinephrine. Skins were darkened by MSH (3 X 10"°
g/ml) for 45 min and then melatonin was added
at 10-' (•), 10-5 ( • ) , or 10-° M ( • ) to the
skins of an experimental group. Norepinephrine
(10-° M) was added to a control group (O) of
skins at 60 min. Each point on the graph is the
mean of 16 measurements of reflectance. Standard
error of the mean is indicated by vertical lines.
COLOR CHANGES IN REPTILES
495
and remains so, irrespective of illumination. At 40°C Anolis becomes green and
remains so, regardless of illumination. At
intermediate temperatures, 20-35°C, the
nature of the illumination is the controlling factor in determining chromatic responses. The rate of color change at intermediate temperatures is still influenced
by the temperature, increasing with higher
temperatures and decreasing at colder
temperatures.
In vitro, Smith (1929) obtained data
similar to those of Parker and Starratt
(1904) indicating a direct effect of temperature on melanophoric responses of A.
ecjiiestris. We have studied the effect of
temperature on the melanophores of A.
carolinensis in vitro, and confirmed the
findings of Parker and Starratt and of
Smith that high temperatures cause lightening of skins and low temperatures darken them (Fig. 4). In addition, we observed
the effects of temperature on melanophoric responses to MSH and to norepinephrine, thus providing a more direct
physiological basis for the changes in color
reported by Parker and Starratt. The rate
of response of skins pre-incubated at 4°C
to MSH was considerably reduced, a result
similar to that observed, in vitro, for skins
of R. pipiens (Shizume, Lerner, and
Fitzpatrick, 1954). In addition, cold temperatures almost totally inhibited the
lightening response of skins to norepinephrine (Fig. 4). Although catecholamines
do not play any role in the normal paling
response to a light background, there is a
distinct possibility that excitement-pallor
in response to stimulation by catecholamine (stress) might be so affected.
Although Hadley (1928) and Smith
(1929) demonstrated a maximal dispersion
of melanin granules by light acting on
melanophores of A. equestris in vitro, only
a minimal effect could be demonstrated
by Hadley (1931) or Kleinholz (19386)
for
A. carolinensis. In our experiments on
DIRECT RESPONSES OF MELANOPHORES TO
A.
carolinensis, utilizing in vitro reflecTEMPERATURE AND TO ILLUMINATION
tance methods we have noted a definite
Parker and Starratt (1904) found that though minimal darkening in response to
Anolis cnralinensis becomes brown at 10°C sunlight and a reversal (lightening) when
suggests that melanin granules aggregate
when circulating pituitary chromatophorotropin is absent.
In larval amphibians the body-lightening response in the dark (Bagnara, I960,
1963, 1966) has been considered to come
from the release of melatonin by the pineal
and/or adjacent area of the brain with a
subsequent direct aggregating action of
this agent on the melanin granules of dermal melanophores (Bagnara, 1964). In
adult R. pipiens, melatonin (Hadley,
1966; Kastin and Schally, 1966) or pineal
extracts (McCord and Allen, 1917) have
only a minimal lightening effect on darkadapted (Hadley, 1966) or hypothalamiclesioned frogs (Kastin and Schally, 1966).
Similarly, in vitro, melatonin has only a
very minimal and transient aggregating
effect on MSH-dispersed melanin granules within melanophores of R. pipiens
(Hadley and Bagnara, 1969), contrary to
the reported effectiveness of this agent
(Lande and Lerner, 1967). In Anolis, melatonin has only a minimal and inconsistent lightening effect in vitro on MSHdarkened skins (Fig. 3). Thus, there are
presently no experimental data to support
a view that the pineal or a pineal hormone, possibly melatonin, plays any role in
chromatic regulation in Anolis.
The suggestion by Kleinholz (1938a)
that integumental photoreceptors reflexly
regulate the release of a hypophyseal chromatophorotropin responsible for controlling melanophoric responses is the only
present explanation for the chromatic responses of blinded lizards. It is difficult to
reconcile this hypothesis, however, with
the demonstration by Kleinholz (1938a)
that lizards still are able to pale under
conditions of darkness after spinal transection. The physiological basis for the paling
response of blinded lizards (Anolis) in
darkness obviously needs further study.
496
-
MAC E. HADLEY AND JOEL M. GOLDMAN
80
FIG. 4. Demonstration in vitro of the direct effect
of temperature on responses of melanophores in
Anolis skins. At time zero, one group ( • ) of skins
was subjected to a warm (50°C) temperature
while another group ( • ) was subjected to cold
(4°C). At 60 min, MSH (3 X 10-* g/ml) wai
added to the cold skins and also to a control
(24°C) group (O) of skins in Ringer's solution. At
that same time a second group (•) of control
skins in Ringer's solution was subjected to a warm
(50°C) temperature. At 120 min, norepinephrine
was added to both groups of MSH-darkened skins.
Each point on the graph is the mean of eight
measurements of reflectance. Vertical lines indicate
the standard error.
skins are placed in darkness. These responses to the presence or absence of illumination can be separated from any experimental differences related to temperature.
In these experiments, the temperature of
the Ringer's solution became warmer (31°
C) under strong sunlight than in darkness
or under fluorescent lighting (24°C), conditions which cause the skins to respond in
the opposite direction (Fig. 4).
"EXCITEMENT-PALLOR" AND "EXCITEMENTDARKENING"
Epinephrine and norepinephrine are
potent agents for aggregating the melanin
granules of vertebrate melanophores. In
vitro, epinephrine aggregates melanin
granules within teleostean (Fundulus
heteroditus; Spaeth, 1916), amphibian
(R. pipiens; Wright, 1955) and reptilian
(A. carolinensis; Horowitz, 1958) melanophores. Hadley (1931) noted, however,
that epinephrine maximally disperses mel-
anin granules within melanophores of A.
iodurus. Kleinholz (1938&) observed that
electrical stimulation of dark-colored individuals of A. carolinensis caused a paling
of the lizards followed by a mottled pattern from dispersion of melanin granules
within localized clusters of melanophores,
the postorbital patch being particularly
noticeable. Similar results followed electrical stimulation of hypophysectomized lizards. Injection of epinephrine mimicked
the mottled condition, and isolated skins
also mottled in response to epinephrine.
Further experiments provided evidence
that epinephrine secreted from the adre. nals regulated this response. Waring (1963)
evaluated these results and concluded that
epinephrine
overrides
the melanindispersing effect of MSH on the melanophores so that melanin granules aggregate.
Then, in localized areas of the integument, epinephrine induces dispersion of
melanin granules within a few melanophores.
We have investigated further the possibility that epinephrine causes both aggregation and dispersion of melanin granules
by studying the response of Anolis skins to
a number of sympathomimetic agents.
Epinephrine (E) lightens or darkens Anolis skins depending on the concentration
used (Fig. 5). Phenylephrine (PE) always
lightens skins, whereas isoproterenol (ISO)
always darkens them. Norepinephrine (NE)
usually lightens skins, but in some experiments darkens them. Whether skins
lighten or darken in response to either
epinephrine or norepinephrine depends
on the initial state of the melanin granules. If skins are maximally light to begin with, due to complete aggregation of
melanin granules within the melanophores, then the skins usually darken in
response to these catecholamines. If, however, skins are initially somewhat dark, due
to a slight dispersion of melanin granules
within the melanophores, then the skins
usually lighten in response to these agents.
As correctly concluded by Horowitz
(1958), whether skins will lighten or darken depends upon the summation of both
497
COLOR CHANGES IN REPTILES
o
z
no
I0o
90
80
_L
15
_L
30
0
15
30
45
TIME, minutes
FIG. 5. A. Comparative response in vitro of Anolis
carolinensis skins to sympathomimetic agents.
Phenylephrine (•), norepinephrine (A), epinephrine (A), and isoproterenol ( • ) were each
added (10"" M) to a group of skins. One group of
skins (O) was maintained as a control in
Ringer's solution. B. In the second experiment, the
sympathomimetic agents were added to give a final
concentration of 10"4 M. Each point on the graphs
is the mean of the reflectance measurements
from the eight skins in the group.
aggregation or dispersion of melanin granules within individual melanophores.
The demonstration that PE, a specific
agonist of alpha adrenergic receptors, always lightens skins is strong evidence that
aggregation of melanin granules is mediated through alpha adrenergic receptors
possessed by the melanophores. In contrast,
dispersion of melanin granules by the
rather specific beta adrenergic receptor agonist, ISO, would be regulated through
beta adrenergic receptors possessed by the
melanophores. The order of relative effectiveness of these sympathomimetic amines
in lightening skins is PE> NE> E > ISO,
which is the reverse order of relative effectiveness of these agonists in darkening the
skins. These rankings are characteristic for
responses mediated by alpha and beta
adrenergic receptors, respectively.
Although the characterization of adrenergic receptors by their physiological response was demonstrated, one can distinguish between alpha and beta adrenergic
receptors much more clearly by using
adrenergic blocking agents (Nickerson,
1967). Pre-incubating Anolis skins in ei-
ther of the alpha adrenergic blocking
agents (Dibenamine or phentolamine)
prevents both norepinephrine and epinephrine from lightening the skins and converts their effects into an "epinephrine
(and norepinephrine) reversal" (Dale,
1906)—an intense darkening of skins resulting from maximal dispersion of melanin
granules within melanophores (Table 1).
After such blockade, the agonists can still
be ranked as to their ability to darken
skins in the following order of relative
effectiveness: ISO>E>NE>PE, thus clearly indicating a role for the beta adrenergic
receptor in regulating the dispersion of
melanin granules.
Pre-incubating skins in beta adrenergic
blocking agents, dichloroisoproterenol (or
propranolol), prevents dispersion of melanin granules in response to isoproterenol
or other sympathomimetic stimulants
(Table 1) and demonstrates that beta
adrenergic receptors of melanophores regTABLK 1. Effect of adrenergic flocking agents on
movement of melanin granules within Anolis melanophores in response to sympathomimetic stimulation.
Experiment
A.
(8)
(8)
(8)
(8)
(8)
(8)
B. (8)
(8)
(8)
(8)
(8)
(8)
C. (7)
(8)
(7)
(8)
Treatment"
1.
2.
3.
4.
5.
6.
1.
2.
3.
4.
5.
6.
1.
2.
3.
4.
% Change in
reflectance*
Dibenamine + ISO
—28 ± 3.31
Dibenamine -f E
—17 ± 2.45
Dibenamine + NB
— 8 ± 1.41
Dibenamine + PE
+ 3 ± 1.04
Einger's
+ ISO
—20 ± 3.11
Einger's
— 1 ± 0.90
Phentolamine + ISO —27 ± 0.98
—27 ± 2.42
Phentolamine -j- E
Phentolamine + NE —13 ± 1.53
Phentolamine + PE + 3 ± 2.89
Einger's
+ ISO —22 ± 2.22
Einger's
+E
—11 ± 1.93
Dibenamine + ISO
—29 ± 3.01
—16 ± 1.48
Dibenamine -j- NE
DCI
+ ISO
— 2 ± 2.40
DCI
-f NE
+ 4 ± 0.95
•Concentrations: Dibenamine, 2 X 10" 5 M; phentolamine, 1 X 10~* M; DCI, dichloroisoproterenol,
agonists: 1 X 10"* M in A
2 X 1Q-* M; adrenergic
and B, 1 X 10""5 M in C. Numbers in parentheses
indicate the number of skins in the group.
* Values are means ± S.E. Mean values represent
the maximal responses within 15 min after adding
the adrenergic agonists. For the control group (in
Einger's solution), the value is the response during
the same time period. ( + ) indicates skin-lightening; (—) indicates darkening of the skins.
498
MAC E. HADLEY AND JOEL M. GOLDMAN
overriding control of these receptors over
the beta receptors also present. The fact
that skins darken in response to ISO indicates the probable presence of some melanophores possessing only beta adrenergic
receptors, or beta receptors which override the action of alpha receptors. Microscopic examination of skins darkened by
ISO did indeed reveal that some, not all,
melanophores had responded to this
catecholamine by dispersing their contained melanin granules (Fig. 7). Skins
pre-incubated in Dibenamine (or phenErgotamine
+ MSH + NE
tolamine or ergotamine) become darker
after adding ISO than do the control skins
30
45
60
in Ringer's solution. Melanin granules
T I M E , minutes
all melanophores become dispersed.
within
FIG. 6. Demonstration in vitro o£ alpha adrenergic
The fact that NE, and E, but not PE, now
receptors mediating aggregation of melanin granules within Anolis melanophores. In one expericause intense darkening of skins, rather
ment, one group of skins (O — O) was incubated
than lightening, after alpha adrenergic
in ergotamine for 30 min, while another group
blockade demonstrates that this reversal by
( • — • ) remained in Ringer's solution as a conepinephrine and norepinephrine comes
trol group; In another experiment two groups
( • — • and O — 0) of skins were incubated with from stimulation of beta adrenergic recepdichloroisoproterenol (DCI) for 30 min. MSH was
tors possessed by the majority of the poputhen added to all four groups. At 60 min, norepilation
of melanophores. These beta recepnephrine was added to the skins of all four
groups except one (O — O) which remained un- tors have been masked heretofore by the
treated as a control. Each point on the graph is the
alpha receptors of the melanophores.
mean of eight measurements of reflectance.
The observation that there is a mosaic
Vertical lines represent the standard error of the
population of melanophores, some possessmean.
ing both alpha and beta adrenergic recepulate dispersion of melanin granules in re- tors and others possibly possessing only
sponse to sympathomimetic stimulation. beta receptors, explains in part why
Skins darkened by MSH lighten with sym- NE, and especially E, can cause both lightpathomimetic stimulation, and the order ening or darkening of skins. If skins are
of response, again, is characteristic of an maximally light in Ringer's solution at the
alpha adrenergic-mediated response. That beginning of the experiment there is little
alpha adrenergic receptors of Anolis me- potential alpha adrenergic response (aglanophores control aggregation of melanin gregation of melanin granules) left to progranules after sympathomimetic stimula- duce lightening, but there is a population
tion is clearly demonstrated when ergota- of melanophores possessing beta adrenergic
mine is shown to block the response (Fig. receptors that does respond to these agents,
6). Beta adrenergic blockade, however, especially E, bringing about darkening
does not block aggregation of melanin of the skin by dispersing melanin grangranules and, in fact, potentiates the re- ules. When skins are darker (for some
sponse. Neither ergotamine nor dichloro- undetermined reason), as measured phoisoproterenol inhibits dispersion of mela- tometrically at the beginning of the exnin granules in response to MSH.
periment, then there is a residual alpha
It is well known that many sympathetic adrenergic response that is initiated by addeffector cells possessing both alpha and beta ing sympathomimetic agents. Whether
adrenergic receptors respond only to stim- the skins lighten or darken depends on the
ulation of alpha receptors due to the relative proportion of cells wherein mel-
COLOR CHANGES IN REPTILES
499
some workers (Sand, 1935) have referred
to as "excitement-darkening." Similarly,
stimulation of the alpha adrenergic receptors of melanophores of dark lizards leads
to a rapid lightening which results in what
is usually referred to as "excitementpallor."
These data also reveal that catecholamines have direct effects on melanophores.
Although Kleinholz indicated a role for
adrenal regulation of color change under
conditions of stress, he considered that the
paling came from vasoconstriction which
prevented MSH from reaching the melanophores. Such a suggestion (Waring, 1963)
is difficult to accept if denervated areas of
the skin, as demonstrated by Kleinholz,
also respond to such stimulation. Also,
Kleinholz ascribed the mottled pattern to
adrenal secretion of epinephrine, but he
%^M^^^^^m* failed to state that this was a direct action
of epinephrine on the melanophores. It is
FIG. 7. Photomicrograph of integumental melanoapparent that his evaluations of the effects
phores of Anolis. Isoproterenol disperses melanin
of epinephrine on changes of color in
granules within certain melanophores, particularly
those comprising the post-orbital patches (an edge Anolis were determined by a desire to esof one is depicted at the top o£ the photo). Other
tablish a unihumoral regulation for meladjacent melanophores remain apparently unreanophores as opposed to the bihumoral
sponsive to such stimulation. The physiological
theory then being formulated by Hogben
basis for these differential responses of melano(1936).
phores probably resides in the nature of adrenergic
receptors possessed by the individual melanoThe present experiments establish that
phores. X 400.
catecholamines directly stimulate moveanin granules are becoming either aggre- ment of melanin granules within melanogated or dispersed. A similar suggestion phores and thus regulate chromatic rewas originally made by Horowitz (1958) sponses under conditions of stress. These
agents, however, as suggested by Kleinholz
working with A. carolinensis.
(1938fc), play no role in either the normal
These results clearly indicate that beta paling response to a light-colored backadrenergic receptors are responsible for ground or the paling response to darkness.
producing the mottled pattern of A. carolinensis in vitro in response to epinephRECEPTORS, CYCLIC 3 ' , 5'-AMP, AND THE
rine, or in vivo when the adrenals are
REGULATION OF MELANOPHORIC
stimulated to release their catecholamines.
RESPONSES
From these data one might expect that
beta but not alpha adrenergic receptors
Methylxanthines such as caffeine (Novaare possessed by melanophores of A. iodu- les, 1959; Wright and Lerner, 1960) and
rus, because maximal dispersion of mela- theophylline (Dikstein, Hirshbein, and
nin granules within all melanophores takes Sulman, 1963: Hadley and Bagnara, 1969)
place in response to stimulation by epi- darken frog skins by dispersing melanin
nephrine (Hadley, 1931). Stimulation of the granules within melanophores. These
beta adrenergic receptors of melanophores agents also darken Anolis skin (Fig. 8).
provides an adequate explanation for what Theophylline is slightly more effective
500
MAC E. HADLEY AND JOEL M. GOLDMAN
yThtob'omint
FIG. 8. Darkening effect of methylxanthines on
Anolis skins in vitro. Skins were placed in Ringer's
solution containing either theobromine
(•),
caffeine (O), or theophylline ( • ) at 5 X 10"3 M
concentrations. At 30 min, norepinephrine (lO"8
M) was added to each group. At 60 min, the
skins in each group were rinsed in Ringer's
solution several times to demonstrate their ability
to relighten. Each point on the graph is the mean
of eight measurements of reflectance. Standard errors are indicated by vertical lines.
than caffeine, and both of these agents are
much more effective than theobromine.
Theophylline and caffeine maximally
darken lizard skins. Neither epinephrine
or norepinephrine relightens these skins although they do lighten and return to their
original reflectance values when washed
repeatedly in fresh Ringer's solution. If
skins are submaximally darkened by MSH
or theophylline the addition of epinephrine or norepinephrine rapidly reverses
the effect of MSH, but, in contrast, causes
a further increase in darkening over that
induced by theophylline (or caffeine)
(Fig. 9).
It has been suggested that hormones,
"first messengers" (Sutherland, Oye, and
Butcher, 1965) exert their effects on cells
by stimulating the intracellular increase of
a "second messenger", cyclic 3', 5'-adenosine monophosphate (cyclic 3', 5'-AMP).
Methylxanthines mimic the action of hormones by inhibiting cyclic 3', 5'-AMP
phosphodiesterase (Sutherland and Rail,
1958) which apparently regulates the intracellular level of cyclic 3', 5'-AMP.
Brodie, et al. (1966) found that adipose
tissue in vitro responds maximally (as
measured by release of glycerol) to high
concentrations of theophylline (5 X 10~3
M), and the response could not be increased by further adding of cyclic 3',
5'-AMP. The observation that we could
not increase darkening of Anolis skins, already maximally darkened by xanthines,
by adding catecholamines is quite analogous to this experiment. Adipose tissue incubated with minimal amounts of theophylline responded to added cyclic AMP
by maximal activation of lipase. Similar
findings have been reported by Vaughan
(1967), wherein it was demonstrated that
caffeine in low concentrations stimulated
release of glycerol which could then be
further increased by adding a small
amount of epinephrine. Similarly, whenever we induce only minimal darkening of
skins by methylxanthines, adding catecholamines darkens them further. We
consider this increased darkening good evidence for a catecholamine-induced increase in intracellular levels of cyclic 3',
5'-AMP.
30
45
60
T I M E , minutes
75
FIG. 9. Comparative response in vitro of MSHdarkened and theophylline-darkened Anolis skins
to epinephrine. Two groups ( • and fj) were
submaximally darkened by theophylline (10"» M)
for 30 min. Then, MSH was added to a control
group (•) of skins in Ringer's solution and to
one of the theophylline-darkened group ( • )
of skins. At 60 min, epinephrine (10"° M) was
added to the skins of each group and also to the
skins of a control group (O) in Ringer's solution.
Each point on the graph is the mean of eight
reflectance reading. Vertical lines indicate the standard error.
501
COLOR CHANCES IN REPTILES
r
"-OMSH + NE
50
15
30
45
60
75
90
105
TIME, minutes
FIG. 10. Comparative response in vitro of Anolis
melanophores to cyclic 3', 5'-AMP (cAMP) and
dibutyryl cyclic 3', 5'-AMP (DBcAMP). In each
experiment (A and B), one group of skins was
incubated for 75 min in either cyclic 3', 5'-AMP
(•) or dibutyryl cyclic 3', 5'-AMP ( • ) while
one group (O) of skins was maintained in
Ringer's solution as controls. The same solutions
of cyclic 3', 5'-AMP and dibutyryl cyclic 3',
5'-AMP were used in both experiments A and B.
MSH was then added to the controls. In the second
experiment (B), norepinephrine (1O"0 M) was
added to the skins of each experimental group at
90 min. Each point on the graph is the mean of
eight measurements of reflectance.
Cyclic AMP itself will not darken Anolis
skins. There are several processes in
which cyclic 3', 5'-AMP is believed to play
a role but where adding exogenous cyclic
3', 5'-AMP has little or no effect. This is
considered to be due to the impermeability
of membranes to the nucleotide together
with its rapid inactivation by phosphodiesterase. Bitensky and Burstein (1965) and
Novaks and Davis (1967) found that cyclic 3', 5'-AMP darkens the skins of Rana
pipiens and considered this evidence that
the first messenger-second messenger hypothesis could account for melanophoric
responses to MSH.
Although MSH and methylxanthines
cause a rapid and maximal darkening of
Anolis skins, cyclic 3', 5'-AMP rapidly
lightens them (Fig. 10A), the magnitude of
the response depending upon the initial
degree of lightness. The dibutyryl derivative of cyclic 3', 5'-AMP, however, causes
a rapid and maximal darkening of Anolis
skins. It has been suggested that the effectiveness of this derivative is due to its in-
creased penetration into cells and the inability of phosphodiesterase to then inactivate it. Dibutyryl cyclic 3', 5'-AMP can
be used a number of times without loss of
activity suggesting that it is not inactivated
(Fig. 10B). Cyclic 3', 5'-AMP, on the
other hand, rapidly loses its aggregating
effect on melanin granules. If MSH darkens
skins by increasing intracellular levels of
cyclic 3', 5'-AMP within melanophores,
then, aggregation of melanin granules in
response to catecholamines may involve an
activation of phosphodiesterase, resulting
in a rapid degradation of cyclic 3', 5'-AMP.
The inability of norepinephrine to reverse
the darkening produced by dibutyryl cyclic
3', 5'-AMP may relate to the fact that the
phosphodiesterase activated by the catecholamine is without effect on the dibutyryl cyclic nucleotide.
We are presently unable to account for
aggregation of melanin granules in response to cyclic 3', 5'-AMP. Our preparations of cyclic 3', 5'-AMP will disperse
melanin granules within melanophores of
502
MAC E. HADLEY AND JOEL M. GOLD>TAN
FIG. 11. Comparative response in vitro of skins of
Anolis carolinenis and Rana pipiens to cyclic 3',
s'-AMP. One group of Anolis (•) and one group
of Rana ( # ) skins were incubated in cyclic 3',
y'-AMT for 90 min. MSH (3 X 10"8 g/ml) was
then added to each group to demonstrate both the
viability and maximal potential darkening response
of these skins. One group of Auolis (QJ) and
one group of Rana (O) skins were maintained as
controls in Ringer's solution. Each point on the
graph is the mean of eight measurements of reflectance. Vertical lines represent the standard error of
the mean.
stimulation would then involve a decrease
in the intracellular level of this cyclic nucleotide. Although it is well documented
that levels of cyclic 3', o'-AMP are mediated through adrenergic receptors under
sympathomimetic stimulation, there is no
information on the "receptors" involved in
dispersion of melanin granules by MSH.
MSH does not apparently mediate its dispersing effect through the beta adrenergic
receptor, for although blockade of this receptor blocks a subsequent response to stimulation by catecholamines, it does not inhibit dispersion of melanin granules by
MSH.
SUMMARY OF EVENTS REGULATING CHROMATIC
RESPONSES IN
Anolis carolinensis
Anolis carolinensis responds to a dark
(light-absorbing) background by releasing
MSH which stimulates dispersion of melanin granules within melanophores leading
to a darkening of the skin. When
Rana pipiens (Fig. 11), as previously
placed
on a light-colored (light-reflecting)
demonstrated by Bitensky and Burstein
background,
MSH is no longer released
(1965) and Novales and Davis (1967).
This response is generally quite minimal, and the animal pales, resulting from aggrebut the response to dibutyryl cyclic 3', gation of melanin granules within melano5'-AMP, as in Anolis, is maximal (Bag- phores. These responses are regulated
through information received through the
nara and Hadley, this symposium).
eyes.
The presence or absence of circulatTurtle and Kipnis (1967) have demonstrated that the intracellular level of cyclic ing MSH adequately accounts for these re?>', o'-AMP is regulated through adrener- sponses. There is no evidence that a secgic receptors. These workers suggested that ond, or lightening hormone ("W" horstimulation of alpha adrenergic receptors mone of Hogben, 1936), such as melatonin,
inhibits, and stimulation of beta adrenergic epinephrine, norepinephrine, or acetylreceptors increases, synthesis o£ cyclic 3', choline regulates the paling response.
Blinded lizards are brown under strong
5'-AMP. There is now a great deal of
evidence to suggest that in those tissues illumination and green in the dark or unwhere beta adrenergic receptors occur der lower light intensities. These responses,
(e.g., the heart and the liver) "these re- too, are apparently regulated by the
ceptors are closely associated with (if not presence or absence of circulating MSH.
an integral component of) the adenyl cy- The only hypothesis to account for chromatic regulation in blinded lizards is the
clase system" (Sutherland, et al., 1968).
The demonstration that stimulation of suggestion of Kleinholz (1938£>) that derthe beta adrenergic receptor results in dis- mal photorecepiors reflexly regulate the repersion of melanin granules suggests, lease of MSH from the hypophysis.
There is no evidence that the pineal
therefore, that this event may indeed involve the intracellular increase in levels of gland, as photoi eceptor and/or endocrine
cyclic 3', 5'-AMP. Aggregation of melanin organ, plays any role in chromatic activity.
granules in response to sympathomimetic The adrenals, however, release catechola-
503
COLOR CHANGES IN REPTILES
mines (probably epinephrine and/or norepinephrine) under conditions of stress.
These catecholamines directly aggregate
melanin granules in integumental melanophores, leading to a paling of brown lizards. They also disperse melanin granules
in some melanophores, giving a mottled
pattern especially prominent as post-orbital
patches. H lizards are initially pale, then
the release of adrenal catecholamines will
also bring into evidence the mottled pattern.
Melanophores are directly responsive to
temperature. Cold darkens and warm temperatures lighten Ai2olis skins in vitro.
Cold greatly slows down the rate of darkening of skins by MSH and inhibits the
aggregation of melanin granules by cate
cholamines. Melanophores respond directly
to illumination: in vitro, under strong
illumination, melanin granules disperse,
whereas they aggregate in the dark. Dispersion of melanin granules within Anolis
melanophores
after
sympathomimetic
stimulation is mediated through beta adrenergic receptors. Aggregation of melanin
granules, on the other hand, after such
stimulation is regulated through alpha
adrenergic receptors. Paling (excitementpallor) after the release of adrenal
catecholamines is regulated, therefore,
through alpha adrenergic receptors possessed by melanophores. Mottling (excitement-darkening) in response to such stimulation is mediated through beta adrenergic
receptors. Jn vitro studies have revealed that
most melanophores possess both alpha and
beta adrenergic receptors. The ability of
Anolis skin to mottle may have its cellular basis in the fact that some melanophores possess only beta adrenergic receptors.
The response of Anolis melanophores to
hormonal stimulation may involve, as for
other endocrine target tissues, changes in
the intracellular levels of cyclic 3', 5'-adenosine monophosphate. An increase in the
intracellular levels of this cyclic nucleotide
is apparently related to dispersion of melanin granules whereas aggregation of the
granules results from lowered intracellular
levels of this nucleotide. Although the beta
adrenergic receptor regulates dispersion
of melanin granules in response to sympathomimetic stimulation, the "receptors"
for MSH-stimulated dispersion of melanin
granules have not been elucidated.
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