The influence of light and dark on
the catecholamine content of the
retina and choroid
Charles W. Nichols, David Jacobowitz, and Marianne Hottenstein
Recent histochemical studies with the use of a fluorescence method to detect catecholaminecontaining neurons have uncovered a system of dopamine-containing amacrine cells as toell as
a plexus of norepinephrine-containing nerve terminals in the choroid. This study examined the
effect of dark and light adaptation on the catecholamine content of the posterior segment of
the eye of the albino guinea pig, rabbit, and rat by quantitative and histochemical means.
In the rat and rabbit, there was a significant increase in the dopamine content with light
adaptation. In the guinea pig, there was an increase, but this was not statistically significant.
A significant increase in the norepinephrine content was observed in the posterior segment of
the guinea pig and rabbit ivith light adaptation. The increase in norepinephrine appeared to
be a direct effect on the choroidal nerve terminals, since with pigmented guinea pigs the
increase was not observed. Likewise, decentralization of the choroid in albino guinea pigs
did not alter the changes seen with light adaptation. Histochemical studies demonstrated a
consistent increase in the fluorescence of the amacrine cells of the rabbit and guinea pig with
light adaptation. It was not possible to show consistent changes in the rat.
A
whereas the sclera is devoid of catecholamine-containing fibers except surrounding
penetrating vessels.4 The function of the
dopamine-containing neurons is unknown.
The present report compares the effect of
light and dark upon the catecholamine
content of the retina and choroid of the
albino rat, guinea pig, and rabbit by histochemical and quantitative methods.
system of intraretinal dopamine-containing neurons located at the junction of
the inner plexiform and inner nuclear
layers of several species was described
with the use of a combination of histochemical and chemical methods for identification and localization of catecholamines.1'3 In addition, a rich plexus of
catecholamine-containing fibers was seen
to surround the vessels of the choroid,3
Methods
Only male subjects were used in this study.
Albino Wistar rats, 200 to 250 grams and 11 to
12 weeks old, albino guinea pigs (Hartley strain),
and pigmented guinea pigs (mongrel strains),
400 to 500 grams and 12 to 16 weeks old, were
light adapted by exposure to daylight for 1 hour
(illumination varied from 330 to 550 lux within
the cages). Albino rabbits (New Zealand white
strain), 3 to 3.3 kilograms and approximately 4
months old, were exposed to a 100 watt incandescent light bulb placed 1% feet above each
From the Departments of Pharmacology and Ophthalmology, School of Medicine, University of
Pennsylvania, Philadelphia, Pa.
Supported by United States Public Health Service Training Grant 2TI-GM-957-05, United
States Public Health Service Research Grant
1-RO1-NB-06707-01, and by Research Career
Program Award 5-K3-NB-13, 935-02 from the
National Institutes of Health.
642
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Volume 6
Number 6
Light and dark on catecholamine
rabbit's head for 1 hour (illumination varied from
275 to 440 lux at eye level). All the animals
were in clear plastic cages without lids or bedding material during light exposure. They were
constantly observed and gentle movement of the
cage was used, when necessary, to assure that
the eyelids remained open and the eyes exposed
to light. All the animals were dark-adapted by
placing them in a photographic darkroom for 24
hours. In addition, albino guinea pigs (350 to
400 grams, 10 to 12 weeks old), whose right
superior cervical preganglionic nerve trunk had
been cut 8 days previously, were light adapted.
Rats and guinea pigs were decapitated, and
rabbits were killed by air embolism. The globes
were rapidly dissected out and the anterior segment removed by cutting about 2 mm. behind
the limbus. The posterior segments of guinea pig
and rabbit eyes were then frozen in liquid
nitrogen. The rat retina was easily separated
from the underlying choroid and was frozen
alone. This procedure for the light-adapted
animals was performed under identical lighting
conditions as those used for light adaptation.
The dark-adapted animals were processed in a
room adjoining the photographic darkroom and
equipped with black shades. No lights were used
and after an adaptation period of about Vz hour,
the investigator could just distinguish necessary
landmarks. The rabbits took longer to kill than
the smaller species. Rabbits were placed in a box
which was sealed so that only the ear was exposed. A second individual injected air intravenously while illuminating the ear vein with a
penlight. The average dissection time, i.e., from
time of death to freezing of the posterior segment, was essentially similar for the light- and
dark-adapted animals; the time for rabbits and
guinea pigs was about 25 seconds for the right
eye and 50 seconds for the left eye. Since the
retina alone was used from rats, this dissection
time increased to 30 seconds for the right and
content of retina
643
65 seconds for the left eye. Histological studies
of the anterior segments from dark-adapted eyes
treated in the above manner verified complete
separation of the anterior from the posterior
segment. All tissues were stored in a frozen state
on dry ice and analyzed for norepinephrine and
dopamine within 24 to 48 hours.
Each experiment consisted of two determinations, one on light-adapted and the other on darkadapted animals. The tissue was weighed and
then for each determination the posterior segments of the eyes from 3 guinea pigs or 2 rabbits
or the retinas from the eyes of 3 rats were
pooled. Dopamine and norepinephrine were determined spectrophotofluorometrically by the
method of Hogans5 as quoted by Jacobowitz and
associates.0 Norepinephrine was activated at 390
m/t, and the emission read at 490 mfi immediately
after processing the tissue. Dopamine was activated at 330 m/t and the emission read at 380
mji at least 20 minutes after the norepinephrine
determinations. The dopamine fluorophore requires 20 minutes to develop and is then stable
for several hours. (Wavelengths given are uncorrected instrumental values.)
For histochemical examination, whole eyes,
light- and dark-adapted as above, were frozen
in isopentane cooled in liquid nitrogen, freezedried in vacuo at -35° C. for 2 days and then
treated with paraformaldehyde gas at 80° C. for
1 hour according to the method of Falck and
associates.7"0 Paraffin sections were cut at 14 m/i
and examined for monoamines with a fluorescence
microscope. All photographs were developed
identically and paired according to equal exposure times for comparison between die fluorescence in dark- and light-adapted animals.
Results
Light adaptation caused an increase in
the catecholamine content of the posterior
Table I. Catecholamine content of the posterior segment of the eye
Dopamine
(ng./posterior segment)
Subject
Rabbit ( 3 ) f
Rat (5)t
Guinea pig (9)
Guinea pig (2) pigmented
Guinea pig (2) preganglionic
denervation
Dark
8.8
3.3
3.5
—
Light
13.7
5.2
4.8
—
—
—
Change
+56 p
+57 p
+37 p
—
—
Norepinephrine
(ng./posterior segment)
P*
< 0.05
< 0.02
> 0.05
—
—
Dark
16.5
—
16.2
18.6
—
Light
Change
P
22.7
+38 p < 0.05
—
—
__
19.3
+19 p < 0.01
19.6
+5
—
12.9
(Denervated)
+6
—
13.8
(Normal)
0
Student t test.
fNumber of experiments.
(Retina only.
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644 Nichols, Jacobowitz, and Hottenstein
Investigative Ophthalmology
December 1967
segment of the eye in all the species
studied (Table I). The values are reported
as nanograms (ng.) per posterior segment
primarily because an unknown amount of
vitreous was inevitably included with each
specimen. This caused considerable differences between individual weights, and
made it undesirable to compare values on
a per gram basis. In the albino rabbit, the
mean dopamine content increased from
8.8 to 13.7 ng. per posterior segment. In
the albino rat, only the retina was used
and there was a mean increase in dopamine
from 3.3 to 5.2 ng. per retina. Although
there appeared to be an increase in
dopamine in the albino guinea pig from
3.5 to 4.8 ng. per posterior segment, this
did not attain statistical significance (p
>0.05).
An increase in the norepinephrine of
the posterior segment was found after
light adaptation in the albino rabbit and
guinea pig. In the rabbit, the norepinephrine increased from 16.5 to 22.7 ng. per
posterior segment. In the guinea pig, there
was an increase from 16.2 to 19.3 ng. per
posterior segment. With pigmented guinea
pigs, however, there was no comparable
increase in norepinephrine with light
adaptation. Furthermore, in albino guinea
pigs, cutting the preganglionic trunk to the
right superior cervical ganglion did not
cause any difference between the two
sides in the norepinephrine content of the
posterior segment in response to light
adaptation. The retinas of rats and guinea
pigs were found to contain measurable
quantities only of dopamine. Dopamine
was shown previously to be the major
catecholamine in the rabbit retina.2-3 The
sclera is not innervated by catecholamine
containing nerve fibers, although a few
fibers do penetrate it in conjunction with
blood vessels.'1 Therefore, the changes in
norepinephrine seen above primarily represent changes in its concentration in the
choroid.
With the use of fluorescence microscopy,
it was found that most of the dopaminecontaining cells of the light-adapted retinas
of rabbits and guinea pigs showed an increase in fluorescence intensity (Figs. 1
and 2). Also noticeable is an increase in
intensity of fluorescent fibers in the inner
plexiform layer of the light-adapted retinas.
It was not possible to demonstrate consistent differences in the fluorescing cells
of dark- and light-adapted rat eyes, probably because visual discrimination could
not distinguish an increase over the already
intensely fluorescent cells seen in the darkadapted retina.
Discussion
Laboratory animals, as well as human
beings, have been known to develop an
Fig. 1. Guinea pig retina. A, dark-adapted; B, light-adapted. Increase in intensity of fluorescence with light adaptation is clearly noted in both the amacrine cell body and the nerve
fibers in the inner plexiform layer. (x480.)
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Volume 6
Number G
Light and dark on catecholamine content of retina 645
increased sensitivity to light during chronic
administration of reserpine, which causes
a depletion of catecholamines from nerve
terminals.10 Malmfors,1 therefore, postulated that the photophobia may be due to
the depletion of an inhibitory transmitter
from adrenergic nerves in the retina. A
possible pathway which is depleted by
reserpine has been described in the rat,1
guinea pig,4 and rabbit.3' " This pathway
is considered to be intraretinal and the
cells of origin have been described as
amacrine cells.11 These cells have been
shown to contain dopamine.2'3 A similar
pathway arising from amacrine cells but
containing acetylcholinesterase has also
been demonstrated.12 Whether or not these
two systems are identical is unknown.
The results of these experiments show
that the retinal dopamine content is increased after light adaptation. This could
be due to an increase in storage of the
catecholamine. However, as mentioned
above, reserpine causes depletion of an apparently inhibitory transmitter required for
light adaptation. The increased dopamine
content, therefore, may well be a manifestation of increased synthesis and release.
The values obtained for dopamine in the
rabbit posterior segment are similar to
those reported by Haggendal and Malmfors,2' 3 compared on a nanogram per organ
basis. They report that these dopamine
values are equivalent to 0.1 to 0.2 jag per
gram of retina, wet weight. This latter
value is 10 per cent of that reported by
Drujan and associates.13 However, he fails
to state whether his values are on a wet
or dry tissue basis. In addition, Drujan
reported a decrease of 57 per cent in the
dopamine content of the retina during
light adaptation in the rabbit. This was a
change from 2.202 ± 0.610 to 1.271 ± 0.490
tig per gram. There is no mention of the
statistical significance of this data. Likewise, the intensity of the light used is not
indicated, but the time of exposure was 4
hours as compared to 1 hour in the present
study. It is conceivable, therefore, that with
longer exposures, or a different light intensity, a decrease in the retinal dopamine
content could occur. Moreover, the length
of time required to process the tissue
might be important. Drujan did not indicate the dissection time. Since he removed
the retina, the time for tissue manipulation
was greater than that of the present study.
The enzymes, monoamine oxidase, and
catechol-o-methyl transferase; responsible
for metabolic degradation of catecholamines, are found in the retina and
choroid.34 It has been shown recently that
monoamine oxidase activity in the retina
is increased with light adaptation.15 It is
suggested that the increase in monoamine
oxidase activity may be a reflection of
Fig. 2. Rabbit retina. A, dark-adapted; B, light-adapted. A noticeable increase in fluorescent
nerve fibers in the inner plexifonn layer with light adaptation is evident together with a
moderate rise in the fluorescence of the amacrine cell. (x480.)
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646 Nichols, Jacobowitz, and Hottenstein
a greater demand for metabolic degradation of the increased content of retinal
dopamine after light adaptation. Therefore, increased time for surgical manipulation could possibly be responsible for a
decrease in the retinal dopamine content
in the light adapted eye.
A surprising increase in the norepinephrine content of the posterior segment was
observed with exposure to light. Since
norepinephrine in the posterior segment
is contained primarily in the choroid as
described above, this change probably
represents an increased content of norepinephrine in the choroidal nerve terminals. Two attempts were made to define
the mechanism of this increase. First, in
pigmented animals, most of the light would
not reach the choroid, thus eliminating
any direct effect of light on nerve terminals. The adrenergic nerves to the choroid
emanate from the superior cervical ganglion
since ganglionectomy causes depletion of
catecholamines in this region.3 If the stimulus to an increase in norepinephrine was
reflexly transmitted through the superior
cervical ganglion, one would see an increase in the light-adapted pigmented
animals. However, no appreciable change
in the norepinephrine content was seen
between the light- and dark-adapted pigmented animals, suggesting that a direct
effect on the choroidal nerves may be
involved. Second, with decentralization of
the superior cervical ganglion prior to light
adaptation, a centrally mediated reflex
would affect only the intact side. The
values, however, for both sides were not
appreciably different. These factors suggest that light may act directly on the
nerve terminals to cause an increase in
choroidal norepinephrine synthesis. Similarly, increased light impinging on the eye
may serve to stimulate the synthesis of
dopamine within the amacrine cells of the
retina.
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