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Light microscopy of the pineal organ of two primitive lizards Platyurus platyurus and Hemidactylus frenatus.

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THE ANA’I’OMICAL RECORD 206283-288 (1983)
Light Microscopy of the Pineal Organ of Two Primitive
Lizards, Platyurus platyurus and Hemidactylus frenatus
VERNON L YEAGER, JOHN J TAYLOR, AND PING LUNG CHANG
of Medicine, St LOULS,
Department ofAnatorny, St Louis IJnzoers~tvSLhool
M O 63104‘
A number of structures, such as paraphysis, dorsal sac, parapineal and pineal organ, may develop in the epithalamic region
of the brain. While nearly all parts are present in some vertebrates, they may all be rudimentary or lacking in others. Interestingly,
the variations do not necessarily follow phylogenic classifications. For instance, the pineal organ is said to be absent in such diverse
forms as hagfish (Myxine glutinosa), rays
(Torpedo ocellata),crocodiles (Crocodilia),certain owls (Strigiformes). armadillos (Dasypus
vellosus), anteaters (Myrmecephagia ,jubota),
dolphins (Delphinus longirostris),and whales
(Balaenoptera borealis) (Oksche, 1965; Renzoni, 1968). A great diversity in morphology
appears among reptiles; where all parts of
the complex are said to be absent in crocodiles (Oksche, 19651, only the proximal portion of the pineal organ is present in turtles
(Hoffman, 1970; Vivien-Roels, 1969), most
parts of the pineal complex are present in the
iguana (Tilney and Warren, 19191, and in
snakes, the pineal resembles the solid parenchymal organ of certain mammals (Hoffman,
1970; Vivien, 1964).
Morphological as well as electrophysiological evidence of a photoreceptive function of
the pineal has been provided for fish
(Breucker and Horstmann, 1965; Rudeberg,
1971; Dodt, 1963), amphibians (Van de Kamer, 1965; Oksche and von Harnack, 1963;
Dodt and Jacobson, 1963), and reptiles
(Oksche and Kirschstein, 1968; Steyn, 1960;
Miller and Wolbarsht, 1962; Hamasaki and
Dodt, 1969).In 1970, Reiter stated, “It is with
confidence that scientists now assert that the
pineal, in all classes of vertebrates, is capable of modulating endocrine activity. . . .”
A light microscopic study of the pineal region of two closely related primitive lizards,
Platyurus platyurus and Hemidactylus frenatus, suggests that it is a very simple transitional form and that it may be useful in
understanding how the transition from photoreceptor to endocrine function occurred.
(c,
1983 ALAN R LISS, INC
MATERIALS AND METHODS
Two species of gecko, Platyurus platyurus
and Hemidactylus frenatus, were identified
on the basis of descriptions by Taylor (1963)
and used for this study. These lizards were
caught in Bangkok, Thailand, and either immediately killed or kept in screen cages in
the laboratory. Those which were kept were
fed cooked rice, mosquitos, and water ad libitum. The animals varied in age from newly
hatched with a snout-vent length of 18 mm
to older animals with snout-vent lengths up
t o 57 mm.
For euthanasia, the lizards were placed in
a refrigerator (2-4°C) until a state of hypothermia existed in which no reflexes could be
elicted. For some specimens, the head was
removed, skinned, and fixed by submersion
in buffered neutral 10% formalin. Other
specimens were perfused through the heart
ventricle with a modified Ringer’s or 0.75%
saline solution to which a small amount of
heparin was added, followed by 3% paraformaldehyde or 3% glutaraldehyde in phosphate buffer adjusted to pH 7.2. Perfusion
was followed by submersion in the same fixative for 2 hours to 2 days. Some of the brains
were then removed and placed in wash buffer.
After fixation, some whole heads were decalcified in 10% formic acid, embedded in
paraffin, and serially sectioned in 8 pm in
sagittal or transverse planes while other
heads were decalcified in 18% EDTA in 4%
buffered (pH 7.2) formaldehyde and embedded in JB-4 embedding mixture (Polysciences, Inc.) for sectioning at 2-3 pm. Tissue
sections were stained with luxol fast blue
and cresyl violet, hematoxylin and eosin, or
periodic acid-Schiff (PAS) reagent. Other epiphyses were removed, postosmicated, dehydrated in alcohol and propylene oxide,
embedded in Epon 812, and 0.5 to 1.0-pm
Received February 1,1983; accepted March 22, 1983.
P.-L.C.’s current address is: Department of Anatomy, College
of Medicine, National Taiwan University, Taipei, Taiwan, R.O.C.
284
V.L. YEAGER, J . J . TAYLOR, AND P.-L. CHANG
cies studied; therefore, the following decription applies to both.
The epiphysis was found to be a small saccular organ attached by connective tissue to
the dorsal aspect of the brain between the
habenular and posterior commissures (Figs.
1, 2). No communication could be found between the epiphysis and the third ventricle.
The epiphysis lies between the posterior extremities of the cerebral hemispheres and
the optic tectum. Immediately anterior to the
epiphysis were complex foldings of the paraphysis-dorsal sac complex. The latter complex resembled choroid plexus and communicated with the third ventricle by two
separate but closely related ducts. A large
venous plexus was closely related to the epiphysis.
The epiphysis had a thin connective tissue
capsule which was continuous with the pia
mater of the epithalamus. The capsule consisted of collagen and reticular fibers with
numerous fibroblasts and a few mast cells
and pigment cells. The outer surface of the
capsule had a more regular outline than its
inner surface, which had occasional septa
projecting into small infoldings of the epithelial lining of the epiphysis. Sometimes the
outer surface exhibited a n indentation, suggesting a multivesicular structure. There was
no indication of a connective tissue or fibrous
stroma separating epiphyseal cells.
A few myelinated nerve fibers were seen in
the capsule, but these were rare. No ganglion
cells were seen in the capsule or any other
location in the epiphysis.
Immediately inside the capsule were numerous small blood vessels, most of which
appeared to be capillaries (Figs. 3, 4). The
epithelial cells were separated from the capillaries by a very narrow PAS-positive basement membrane. When there were infoldings
of the epithelium, the connective tissue septa
contained blood vessels also.
The epithelial lining gave the appearance
of a pseudostratified columnar epithelium,
but since the outer limiting cell membranes
were not stained by any of the techniques
used, this could not be established. In most
Fig. 1. Sagittal section of brain showing cerebral
hemisphere (CH), paraphysis (P), dorsal sac (DS), epi-
physis (Et, optic tectum (OT), and cerebellum (C). Paraf
fin section, luxol fast blue stain. X 42.
sections were cut on a n ultramicrotome and
stained with toluidine blue.
RESULTS
No differences were found in the two spe-
LIZARD PINEAL ORGAN
285
Fig. 2. Section of an epiphysis showing its vesicular
structure. The capsule (Ca), numerous blood vessels (arrows), epithelium, and debris in the lumen (L)are clearly
seen. Portions of the paraphysis-dorsal sac complex (PDS) are included. Epon section, toluidine blue stain.
epon sections studied with the light microscope, the epithelium was seen to differ in its
outer, intermediate, and inner zones (Fig. 3).
The outer or basal zone was made up primarily of cytoplasm and intercellular spaces. In
some perfused specimens, these spaces were
enlarged and extended into the other zones.
Specimens fixed by immersion had spaces
the size of capillary lumina or smaller. None
of the spaces had distinguishable linings.
Very few nuclei were found in this zone. The
blood vessels were located at the base of this
zone so that three sides of the capillaries
were related to the epithelium and one side
to the connective tissue capsule (Fig. 4).
The intermediate zone was about twice the
thickness of the outer or basal zone and consisted mostly of closely packed nuclei. These
nuclei were round, oval or tear-drop shaped.
Typically there were prominent nucleoli
within the pale nucleoplasm. Chromatin was
found adjacent t o the nuclear membrane. Intercellular spaces were not evident in this
layer in tissues fixed by immersion. The cytoplasm was scanty in this zone and not remarkable. Occasional very dense bodies were
noted in the supranuclear cytoplasm of this
zone, but these bodies were more characteristic of the inner zone. Most of the nuclei and
cells of this zone appeared t o be similar, but
occasional very dense cells were noted (Fig.
5 ) . In some of these cells, only the nucleus
was more intensely stained, but in others,
both the cytoplasm and nucleus were stained
more deeply than the typical cell.
The inner zone adjacent to the lumen was
the thinnest zone. It had nuclei, but not in
the numbers seen in the intermediate zone.
X
215.
Fig. 3. Higher magnification of the wall of the epiphysis from a specimen fixed by immersion. Note t h e
capsule (Ca), blood vessels (arrows), intercellular spaces
(I) in the outer zone, packed tear-shape6 nuclei in the
Most of the nuclei were somewhat smaller
than those of the intermediate zone and the
chromatin was more clumped. Many of these
had nuclear bodies or inclusions (Fig. 4).In
some areas of some of the epon sections, a
series of stained dots appeared parallel to the
lumen in the apical portions of the cells (Fig.
3). In obliquely cut epithelia, a lattice pattern indicated that these probably represented terminal bars or cell junctions. The
very dense bodies described in the intermediate zone were especially common in this
zone. In some specimens, there appeared to
be one of these bodies in every cell. The bodies were stained by toluidine blue but not by
hematoxylin or PAS treatment. No detail
could be seen within the bodies even when
lightly stained sections were studied.
The luminal surface of the epithelial cells
was rounded or irregular, and material in
the lumen seemed to indicate that degenerating cells, cell parts, or secretory material
was being extruded into the lumen. A slight
PAS reaction occurred a t the lumen surface
or immediately deep to it. The lumen of the
epiphysis appeared to be an actual fluid-filled
space rather than a potential or artifactual
space.
DISCUSSION
If the region between the habenular and
posterior commissures gives rise to only one
intermediate zone, and the inner zone adjacent to the
lumen (L). The series of small dots in the inner zone
probably represent junctional complexes. Epon section,
toluidine blue stain. x 750.
vesicular structure, as is the case for the two
species of gecko studied in this report, some
problem in identification may occur, since
end-vesicles and proximal portions for both
parapineal organs and pineal organs have
been described for lizards. Tilney and Warren (1919) state that for Gecko versus and
Platydactylus muralis only the epiphysis is
present, whereas for Hemidactylus uerruculatus they mention only a n end-vesicle, and
for Hemidactylus mabouia and Gehyra oceanica they mention a n end-vesicle and a stalk.
It is not clear whether they studied the pineal region of geckos themselves or simply
reviewed the findings of others. Since the
structure described in this report resembles
in position and shape the proximal portion of
the complex in other forms where the endvesicle is also present, and because the proximal portion is the most constant part of the
pineal complex, the present authors believe
it is the proximal portion of the pineal organ
and the homolog of the mammalian epiphysis cerebri. Additional studies, especially
Fig. 5. Specimen showing dense cells. The three zones
are indicated by lines (- - -) near the right side of the
figure. Note also t h e difference in appearance of many
of the nuclei in the inner zone compared to those of the
intermediate zone. Epon section, toluidine blue stain.
x 980.
Fig. 4. Higher magnification from a specimen fixed
by perfusion. Note the dilated blood vessels IBV), intercellular spaces (I), supranuclear dense bodies (DB), nu-
cleus with inclusion body (arrow), and clublike cell
processes projecting into t h e lumen (L). Epon section,
toluidine blue stain. x 1830.
5
288
V.L. YEAGER, J.J.TAYLOR, AND P.-L. CHANG
of embryological specimens, are needed to
definitely prove its identity.
To question whether the epiphysis of these
geckos is functioning as a photoreceptor organ, a n endocrine organ, or both cannot be
answered by a light microscopic study. The
cell shape and arrangement is similar to that
found in lower forms in which photoreception
still occurs, but outer segments could not be
identified. In thin plastic sections of the retina of the lateral eye of these lizards, the
outer segments were very apparent, but
nothing comparable was seen in the pineal.
Ganglion cells were never identified, nor was
a pineal nerve found, although two or three
myelinated fibers were seen in or near the
capsule in a few specimens. Pigment cells,
which form a prominent feature of the parietal organ of some reptiles (Tilney and Warren, 1919; Oksche and Kirschstein, 19681,
occurred in very small numbers. Therefore,
morphologic evidence for photoreception was
not convincing.
In a number of saccular epiphyses the lumen communicates with the third ventricle
(Ralph, 1970; Hafeez, 1971; Rudeberg, 1971).
In this study, both the paraphysis and the
dorsal sac were found to have long attenuated ductlike communications with the
third ventricle, but careful examination of 2pm thick serial sections revealed no communication between the ventricular system and
the epiphysis. If the fluid-filled cavity of the
epiphysis receives secretory material, there
does not appear to be a ready exit for it.
A possible route for secretory products, if
they exist, would seem to be the intercellular
spaces in the basal zone adjacent to the capillaries. Quay (1974) has shown by India ink
perfusions that intercellular canaliculi exist
in the rat pineal and he suggests that they
constitute a transport route between pinealocytes and blood vessels. The number and
size of the capillaries suggest that the cells
of the epiphysis in the gecko pineal are metabolically active.
Whether the gecko epiphysis cerebri functions as either a photoreceptor or an endocrine organ could not be established in this
light microscopic study.
ACKNOWLEDGMENTS
The assistance of Mrs. Amara Kumnirdsena Megpaibul and Mrs. Wantanee Trakulrungsi is gratefully acknowledged.
LITERATURE CITED
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