Leucine-enkepholin-like Immunoreacfivity in the
Chicken Retina with a Special Reference to
Its Fine Structures
Ichiro Ishimoro,* Sadao Shiosakaf Yoshiki Shimizu,* Yasuaki Kuwoyomo,* Mosokotsu Fukuda,*
Shinobu lnagaki,j- Hiroshi Takagi,t Mosahiro Sakanaka.j- Arsuko Sasaoka,*
Emiko Senba,t Tadao Sakiyama,* and Mosoya Tohyamat
Leucine-enkephalin-like immunoreactivity (LEI) in the chicken retina was investigated using light
and electron microscopic immunohistochemistry. A flat-mount technique for light microscopy clearly
demonstrated that LEI cells were distributed widely throughout the entire retina, without any differences between the central and peripheral retinal regions. Frozen sections for light microscopy
confirmed the previous findings in pigeons that LEI is localized in amacrine cells. The present electron
microscopic observations confirmed the findings of light microscopy and revealed that LEI terminals
make synaptic contact mainly with vesicle-containing processes that seemed to belong to the amacrine
cells. The present study further suggests that LEI terminals are both pre- and postsynaptic elements
onto vesicle-containing processes mentioned above. Invest Ophthalmol Vis Sci 24:879-885, 1983
Recently, the presence of leucine-enkephalin (LE)
in the retina, particularly in the amacrine cells, has
been demonstrated.1'210131617 However, as to the
functional role of the retinal LE, little is known, although there exist some suggestions.6'717 In order to
explore the functional role of the retinal LE, it seems
necessary to elucidate the fine structure, synaptic contacts, and overall distribution of the LE cells in the
retina. Unfortunately in the previous immunohistochemical studies, it was difficult to determine the
overall distribution of the LE cells because the authors
used frozen sections. However, we recently have suceeded in demonstrating peptide-like immunoreactivity in flat-mounted retinal tissue which make it
possible for us to determine the overall distribution
of LE in the retina. Therefore, in the present study,
we first attempted to describe the overall distribution
of LE-like immunoreactivity (LEI) using flat-mounts.
We then attempted to explore the fine structure of
LEI cells and terminals, little known until now, using
the unlabeled antibody enzyme method of immunocytochemistry.'8
Materials and Methods
Experiments were done on 30 chickens.
Antiserum: Antiserum against LE was produced in
response to a CDI (l-ethyl-(3,3-dimethalminopropyl)-catbodiimide) synthetic LE (Jpn. Peptide Lab.
Co. Ltd) conjugated bovine thyroglobulin (Sigma).
The specificity of LE antiserum was determined by
radioimmunoassay and was shown to cross-react less
than 0.1% with methionine-enkephalin and less than
0.01% with dynorphine and ACTH. There was no
cross-reactivity with endorphins, substance P, somatostatin, neurotensin, cholecystokinin (CCK-8),
LHRH, or TRF. The specificity of immunoreactive
staining also was established by preabsorption of antiserum with excess LE (0.8 ng/vcA).
Tissue preparation and immunohistochemical procedure: Light microscopy: 14 chickens (each of about
50 g body weight) were used. Under nembutal anesthesia (30 mg/kg, ip) each animal was perfused via
its ascending aorta with 30 ml of ice-cold saline followed by Zamboni'sfixative.18In some cases, during
the perfusion the eyes also were infused with the same
fixative. After the perfusion, the eyes were enucleated
immediately and the anterior eye segments were removed. For frozen sections, eye cups were postfixed
for one to two days at 4 C in the same fixative and
then were rinsed for 24 hr in 0.1 M phosphate buffer
containing 30% sucrose. Sections of the retina were
cut on a cryostat either perpendicular to the vertical
axis of the eye at 10 nm or tangential to the retinal
* Department of Ophthalmology, Osaka University Medical
School, and the fDepartment of Neuroanatomy, Institute of
Higher Nervous Activity, Osaka University Medical School, Fukushimaku, Osaka, Japan.
Submitted for publication December 16, 1981.
Reprint requests: Dr. I. Ishimoto, MD, Department of Ophthalmology, Osaka University Medical School, 1-1-50 Fukushima,
Fukushimaku, Osaka (553), Japan.
0146-0404/83/0700/879/$ 1.15 © Association for Research in Vision and Ophthalmology
879
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1983
surface at 10-15 ^m. For retinal flat mounts, eye cups
were prepared as above. Next the pigment epithelium
was separated microsurgically from the retina. The
isolated retina was postfixed in the same fixative for
one to two days and rinsed in 0.1 M phosphate buffer
containing 30% sucrose. Then, the isolated retina was
flat-mounted on a gelatin-coated glass plate with the
vitreal side facing up. All the tissues were subjected
to either the indirect immunofluorescence5 or the
unlabeled antibody-enzyme method of immunohistochemistry.18 The sections first were washed in 0.1
M phosphate buffer saline (PBS) [with or without 10%
goat serum and 4% bovine serum albumin (BSA)] for
10 min at room temperature (15 C) prior to the beginning of the immunohistochemistry. One-half of
the frozen sections and flat-mounts were incubated
in the primary antiserum (which has been pretreated
with thyroglobulin for 45 min) at a dilution of 1:1,000
for 24 hr at 4 C in a humid atmosphere; they then
were rinsed in ice-cold PBS containing 1% Triton X100 for 15 min and then immediately with PBS for
10 min. For the immunofluorescence procedure, the
tissue was incubated with fluorescein isothiocyanate
conjugated antibodies (Miles Co. Ltd.) at a dilution
of 1:50 under the same conditions as described above.
The tissue was washed subsequently in PBS for 10
min at 4 C and mounted in a PBS-glycerine mixture.
For the unlabeled antibody enzyme technique, the
tissue was incubated in goat-anti-rabbit IgG (Miles
Co. Ltd.) at a dilution of 1:50 for 45 min at room
temperature, and washed three times for 15 min in
0.1 M PBS. After being washed, the tissue was incubated in peroxidase-antiperoxidase complex
(DAKO) at a dilution of 1:100 for 45 min at room
temperature, following which it was rinsed three
times, preincubated with 3,3' diaminobenzidine HC1
(DAB) (5 mg/10 ml) for 30 min, and then incubated
for 2 min in DAB (5 mg/10 ml) containing 0.01%
H 2 O 2 . After the reaction was completed, the materials
were washed in 0.1 M PBS, dehydrated, and mounted
in balsam. The remaining tissue was incubated first
in the control LE-preabsorbed seninvand .then, subjected to the immunohistochemical procedures described above.
Electron microscopy: Sixteen chickens (each of
about 50 g body weight) were used for electron mi?
croscopy. Each animal was perfused via its ascending
aorta with 30 ml ice-cold saline, followed by 200 ml
modified Zamboni's solution (0.6% glutaraldehyde,
4% paraformaldehyde, and 0.21% picric acid in 0.1
M phosphate buffer) under nembutal anesthesia (30
mg/kg, ip). During the perfusion, in some cases, the
eyes also were infused with fixative. After perfusion,
the eyes were enucleated immediately and fixed in
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Vol. 24
the same fixative without glutaraldehyde for 24 hr.
After a buffer rinse, the retinas were cut with a Vi-bratome® at 10 ^m. One-half of the sections then
were subjected to the unlabeled antibody enzyme
method of immunohistochemistry described above,
but without using Triton X-100. The other half of
the sections were processed as control. Following the
immunohistochemical processing, the sections were
postfixed in 2% osmium tetroxide (OsO4) for 2 hr at
4 C, dehydrated in graded alcohol, and embedded in
Epon. Serial ultrathin sections were cut and observations were made on both stained (uranyl acetate
and lead citrate according to conventional methods)
and nonstained sections.
Results
I. Control Experiments
The specificity of the immunoreaction was checked
morphologically by comparing sections stained with
LE antiserum and absorbed control serum (the specificity also was checked by radioimmunoassay; see
Materials and Methods). The use of antiserum to LE
absorbed with excess synthetic LE completely abolished immunostaining, indicating that structures
stained by LE antiserum may be considered specifically stained. The specific property of these materials
should be correctly described as LE-like immunoreactivity, but in this study, we will use the simpler
term LEI.
II. Light Microscopic Observations
In the frozen sections, in accordance with previous
studies,1-210'17 numerous LEI cells were observed in
the proximal portion of the inner nuclear layer (INL)
(Figs. 1A and 2). The majority of these LEI cell bodies
were oval and about 7 nm in diameter. No immunoreactive cells were observed in other layers of the
retina. The processes originating from these LEI cells
entered the inner plexiform layer (IPL) where they
arborized, mainly in lamina 3-4, and partly in lamina
1 of (Figs. lAand2C).
In the flat-mounted retina, an overall distribution
of LEI cells was observed (Fig. IB). As shown in Fig.
IB, LEI cells were widely distributed in the entire
retina. The densities of the LEI cells in the central
and peripheral retinal regions were almost the same
(n = 1,600/mm2) and no regional differences in the
distribution of the LEI cells throughout the retina
were detected. Immunoreactive cell perikarya measured in the flat-mounts had a diameter of about 7
fim and the cell-to-cell spacing was approximately
45 /mi.
No. 7
ENKEPHALIN IN THE CHICKEN RETINA / Ishimoro er ol.
881
Fig. 1 A. Fluorescent photomicrograph
showing LEI structures in the chicken retina. Frozen section. LEI cells were located
in the proximal part of the inner nuclear
layer (INL) and processes from these cells
ramify in lamina 1 or laminae 3-4 of the
inner plexiform layer (IPL). B. Fluorescent
photomicrograph showing the distribution
of LEI cells in the retinal region. Flatmount. LEI cells were distributed evenly in
the entire retina, both in the central and
peripheral retinal regions (A, X35O; B,
XI50).
III. Electron Microscopic Observations
Fig. 2C is a bright-field photomicrograph showing
LEI in an Epon-embedded section. In Fig. 2C, as in
Fig. 1A, LEI cells were located in the proximal part
of the INL and LEI fibers were seen mainly in laminae
3-4 and partly in lamina 1. Fig; 2A is an electron
micrograph of the perikaryon and its vitreal process
of the LEI cell shown by the arrow in Fig. 2C. In
agreement with the results from frozen sections, cell
bodies labeled by the LE reaction product were located in the proximal part of the INL (Fig. 2A), and
were oval and about 7 pm in diameter. The nucleus
of these cells was surrounded by a thin rim of cytoplasm (Figs. 2 A and 3), and its surface was smooth
and not indented (Figs. 2 A and 3). In the cytoplasm,
numerous patches of LEI reaction product were identified around the endoplasmic reticulum (Fig. 3). It
is noteworthy that the LEI cell surface were always
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ensheathed by processes of Miiller cells (Figs. 2A
and 3).
The processes from the LEI cells enter the IPL,
being ensheathed by processes of Miiller cells (Figs.
2A and B) and arborize mainly in laminae 3-4 and
partly in lamina 1. In Fig. 2 A, the arborization of LEI
fibers in the lamina 1 is seen. In the ascending processes immunoreactive reaction products were observed along the microtubules (Figs. 2A and B). After
arborization, LEI fibers are made up of varicosities
interconnected by thin portions. These fibers often
lacked the ensheathing Miiller cell processes and
showed direct apposition with neuronal elements.
The varicose terminals in the IPL measured 0.52.5 ftm in diameter and contained numerous synaptic
vesicles (Fig. 4). The fine structure of the LEI fibers
was almost the same throughout the IPL, and no remarkable differences were detected between the LEI
882
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1983
Vol. 24
Fig. 2 A. Electron micrograph of LEl cells and its process. The
LEI cell is located in the proximal part of the INL and its process
ramified in the IPL; here ramification of the LEI fibers in lamina
1 is seen. It should be noted that the LEI cell and its process is
ensheathed by the process of a Muller cells (M) (see also Figs. 2B
and 3). Note that reaction product is seen around the membraneous
structures and microtubules (see also Figs. 2B and 3). Note also
the smooth surface of the nucleus (N) of the LEI celt and the small
amount of its cytoplasm (specifically see Fig. 3) (X7700). B. High
magnification of Fig. 2A. Note that ascending process from LEI
cell is ensheathed by process of Miiller cell and that reaction products are associated with microtubules (XI 7,200). C. Light-field
photomicrograph of LEI cell shown in Figs. 2A and B (arrow).
Epon-embedded materials (X700).
fibers located in laminae 3-4 and 1. The LEI terminals have output synapses of the conventional kind
such as (1) a presynaptic cluster of small synaptic
vesicles close to the presynaptic membrane (Fig. 4B)
and (2) pre- and postsynaptic membrane specialization (Figs. 4A-C) etc. These characteristic features
are very similar to those of amacrine cells shown by
Dowling and Boycott.8 The neruonal elements with
which LEI terminals make synaptic contact are
mainly vesicle-containing processes. These non-labeled processes are likely from amacrine cells, because they always lack synaptic ribbons and the synaptic vesicles in the non-labeled processes are distributed unevenly.7 Thefindingsmentioned above suggest
that LEI terminals are presynaptic in structure. However, it should be noted that LEI processes also look
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like postsynaptic in structure, because the cluster of
vesicles was identified in the unlabeled vesicle-containing processes (Fig. 4B, shown by thin arrow) with
which LEI terminals make synaptic contact as a presynaptic structure and no aggregation of vesicles were
seen in the labeled process opposite the membrane
density (Fig. 4B). These findings suggest that LEI terminals make a reciprocal synapse with non-labeled
vesicle-containing process.
It should be noted that LEI terminals are involved
frequently in complicated neuronal clusters. For example, as shown in Fig. 4C, a single LEI terminal is
involved in synaptic contact with at least three unlabeled terminals, probably from amacrine cells, although in thisfigureit is not clear as to which polarity
these contacts have.
No. 7
883
ENKEPHALIN IN THE CHICKEN RETINA / Ishimoro er ol.
Fig. 3. Electron micrograph showing the
fine structure of LEI cells, from serial sections. Note smooth surface of the nucleus
(N), small amount of cytoplasm, and ensheathment by Muller cell process (M).
Note also that immunoreaction products
are seen around the endoplasmic reticulum
(X20,000).
N
•,t
3
Discussion
Previous immunohistochemical studies12'10'17 using frozen sections have demonstrated that LEI is
localized in the amacrine cells. The present light
microscopic observations using flat-mounts together
with frozen sections have confirmed the previous
findings and further elucidated that LEI cells are distributed widely in the retina, and that no regional
differences were identified. This finding is h> contrast
with the distribution of LEI cells in the pigeon retina,2
in which they are more common in the red fields.
This discrepancy may be attributed to the different
species of birds or different kinds of antisera. Previously, we have reported that in flat mounts, substance
P (SP) cells are localized mainly in the peripheral
retinal region and are few in number in the central
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retinal region.14 These findings strongly suggest that
LE and SP cells in the retina are functionally and
structurally distinct, although, both these peptides are
localized in the amacrine cells. This concept is in
good agreement with the suggestion by Brecha et al 317
that different cell types contain different peptides.
The present study also has demonstrated at the
electron microscopic level that LEI is localized in
amacrine cells. However, although LEI cells possess
many of the well-known characteristics of amacrine
cells,1012 some of their structural features are unusual.
For example, the diameter of the LEI cells is smaller,
the cytoplasm of the LEI cells is much less, and the
surface of the nuclei of LEI cells is smooth and not
indented as in many ordinary amacrines, suggesting
the heterogenous function of the amacrine cells as
already mentioned above. The present electron mi-
884
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1983
jr1
B
Vol. 24
Fig. 4. A and B. Electron micrographs of LEI terminals, from
serial sections. LEI terminals make a synaptic contact with nonlabeled vesicle-filled process which is supposed as a terminal of an
amacrine cells (a), because no synaptic ribbon was identified in the
terminals and synaptic vesicles were distributed unevenly. At the
contact zone, an aggregation of synaptic vesicles in the labeled
terminals (Fig. 4B, indicated by thick arrow) and pre- and postmembraneous thickening (Figs. 4A and B) were seen. Note a cluster
of vesicles in the unlabeled process and that there are no vesicles
in the labeled process opposite the membrane density (thin arrow).
These findings suggest that LEI processes are both pre- and postsynaptic and may constitute reciprocal synapse with vesicle-containing unlabeled processes. C. LEI terminals often make neuronal
cluster. LEI terminals make a synaptic contact simultaneously with
several nonlabeled vesicle-containing processes (A and B, X43,0OO;
C, X40?000).
4c
croscopic study has shown that ascending processes
from LEI cells were ensheathed by processes of Miiller
cells and after arborization, LEI fibers are made up
of varicosities interconnected by thin portions, but
these fiber often lacked the ensheathing Miiller cell
processes, suggesting the areas which lacked the ensheathing Miiller cell process may be an active sites.
The present study has shown that LEI terminals
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make a reciprocal synapse mainly with vesicle-containing processes, but no obvious contact with dendrites were identified. The processes with which LEI
terminals make synaptic contact seem to originate
from amacrine cells, because their synaptic vesicles
are distributed unevenly and no synaptic ribbons
were detected. Some of these amacrine cells may contain GABA, since a recent electrophysiologic study6
No. 7
ENKEPHALIN IN THE CHICKEN RETINA / Ishimoro er ol.
has suggested an axo-axonic contact between LEIand GABA-containing 19 amacrine cells.
Key words: enkephalin, amacrine cells, flat-mounts, fine
structure
10.
11.
Acknowledgments
The authors are grateful to Professor R. Manabe (Department of Ophthalmology) and Professor Y. Shiotani
(Department of Neuroanatomy) for their encouraging advice and supervision during this investigation. The authors
wish to thank Miss T. Tanaka for her excellent phototechniques.
12.
13.
14.
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