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 Department ofAnatorny, St Louis IJnzoers~tv SLhool of Medicine, St LOULS, MO 63104‘

A number of structures, such as para- physis, dorsal sac, parapineal and pineal or- gan, may develop in the epithalamic region of the brain. While nearly all parts are pres- ent in some vertebrates, they may all be ru- dimentary or lacking in others. Interestingly, the variations do not necessarily follow phy- logenic classifications. For instance, the pi- neal organ is said to be absent in such diverse forms as hagfish (Myxine glutinosa), rays (Torpedo ocellata), crocodiles (Crocodilia), cer- tain owls (Strigiformes). armadillos (Dasypus vellosus), anteaters (Myrmecephagia , jubota), dolphins (Delphinus longirostris), and whales (Balaenoptera borealis) (Oksche, 1965; Ren- zoni, 1968). A great diversity in morphology appears among reptiles; where all parts of the complex are said to be absent in croco- diles (Oksche, 19651, only the proximal por- tion 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 paren- chymal organ of certain mammals (Hoffman, 1970; Vivien, 1964).

Morphological as well as electrophysiologi- cal evidence of a photoreceptive function of the pineal has been provided for fish (Breucker and Horstmann, 1965; Rudeberg, 1971; Dodt, 1963), amphibians (Van de Ka- mer, 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 capa- ble of modulating endocrine activity. . . .”

A light microscopic study of the pineal re- gion of two closely related primitive lizards, Platyurus platyurus and Hemidactylus fren- atus, suggests that it is a very simple transi- tional form and that it may be useful in understanding how the transition from pho- toreceptor to endocrine function occurred.

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 im- mediately killed or kept in screen cages in the laboratory. Those which were kept were fed cooked rice, mosquitos, and water ad li- bitum. The animals varied in age from newly hatched with a snout-vent length of 18 mm to older animals with snout-vent lengths up to 57 mm.

For euthanasia, the lizards were placed in a refrigerator (2-4°C) until a state of hypo- thermia 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% parafor- maldehyde or 3% glutaraldehyde in phos- phate buffer adjusted to pH 7.2. Perfusion was followed by submersion in the same fix- ative for 2 hours to 2 days. Some of the brains were then removed and placed in wash buffer.

After fixation, some whole heads were de- calcified 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 embed- ded in JB-4 embedding mixture (Polysci- ences, 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 epi- physes were removed, postosmicated, dehy- drated 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.

(c, 1983 ALAN R LISS, INC

284 V.L. YEAGER, J . J . TAYLOR, AND P.-L. CHANG

sections were cut on an ultramicrotome and stained with toluidine blue.

RESULTS

No differences were found in the two spe- cies studied; therefore, the following decrip- tion applies to both.

The epiphysis was found to be a small sac- cular 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 be- tween the epiphysis and the third ventricle. The epiphysis lies between the posterior ex- tremities of the cerebral hemispheres and the optic tectum. Immediately anterior to the epiphysis were complex foldings of the para- physis-dorsal sac complex. The latter com- plex resembled choroid plexus and com- municated with the third ventricle by two separate but closely related ducts. A large venous plexus was closely related to the epi- physis.

The epiphysis had a thin connective tissue capsule which was continuous with the pia mater of the epithalamus. The capsule con- sisted 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 epithe- lial lining of the epiphysis. Sometimes the outer surface exhibited an indentation, sug- gesting 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 nu- merous small blood vessels, most of which appeared to be capillaries (Figs. 3, 4). The epithelial cells were separated from the cap- illaries by a very narrow PAS-positive base- ment 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 physis (Et, optic tectum (OT), and cerebellum (C). Paraf hemisphere (CH), paraphysis (P), dorsal sac (DS), epi- fin section, luxol fast blue stain. X 42.

LIZARD PINEAL ORGAN 285

Fig. 2. Section of an epiphysis showing its vesicular structure. The capsule (Ca), numerous blood vessels (ar- rows), epithelium, and debris in the lumen (L) are clearly

seen. Portions of the paraphysis-dorsal sac complex (P- DS) are included. Epon section, toluidine blue stain. X 215.

epon sections studied with the light micro- scope, the epithelium was seen to differ in its outer, intermediate, and inner zones (Fig. 3). The outer or basal zone was made up primar- ily 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 con- sisted 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 to the nuclear membrane. In- tercellular spaces were not evident in this layer in tissues fixed by immersion. The cy- toplasm was scanty in this zone and not re- markable. Occasional very dense bodies were noted in the supranuclear cytoplasm of this zone, but these bodies were more character- istic of the inner zone. Most of the nuclei and cells of this zone appeared to 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.

Fig. 3. Higher magnification of the wall of the epi- physis from a specimen fixed by immersion. Note the capsule (Ca), blood vessels (arrows), intercellular spaces (I) in the outer zone, packed tear-shape6 nuclei in the

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.

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 pat- tern indicated that these probably repre- sented terminal bars or cell junctions. The very dense bodies described in the interme- diate zone were especially common in this zone. In some specimens, there appeared to be one of these bodies in every cell. The bod- ies 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 degener- ating cells, cell parts, or secretory material was being extruded into the lumen. A slight PAS reaction occurred at 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

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 War- ren (1919) state that for Gecko versus and Platydactylus muralis only the epiphysis is present, whereas for Hemidactylus uerrucu- latus they mention only an end-vesicle, and for Hemidactylus mabouia and Gehyra ocean- ica they mention an end-vesicle and a stalk. It is not clear whether they studied the pi- neal 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 end- vesicle is also present, and because the prox- imal 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 epi- physis 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 the 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), inter- cellular spaces (I), supranuclear dense bodies (DB), nu-

cleus with inclusion body (arrow), and clublike cell processes projecting into the lumen (L). Epon section, toluidine blue stain. x 1830.

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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 or- gan, an 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 ret- ina 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 parie- tal organ of some reptiles (Tilney and War- ren, 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 lu- men 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 atten- uated ductlike communications with the third ventricle, but careful examination of 2- pm thick serial sections revealed no commu- nication 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 cap- illaries. 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 pineal- ocytes and blood vessels. The number and size of the capillaries suggest that the cells of the epiphysis in the gecko pineal are met- abolically active.

Whether the gecko epiphysis cerebri func- tions as either a photoreceptor or an endo- crine organ could not be established in this light microscopic study.

ACKNOWLEDGMENTS

The assistance of Mrs. Amara Kumnird- sena Megpaibul and Mrs. Wantanee Trakul- rungsi is gratefully acknowledged.

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