Two Components of the Pineal Organ in the Mink (Mustela Vison) : Their Structural Similarity to Submammalian Pineal Complexes and Calcification*

Arch. Histol. Cytol., Vol. 55, No. 5 (1992) p. 477-489

Two Components of the Pineal Organ in the Mink (Mustela vison) : Their Structural Similarity to Submammalian Pineal Complexes and Calcification*

Bela VIGH and Ingeborg VIGH-TEICHMANN

Photoneuroendocrine Laboratory of the Second Department of Anatomy, Histology and Embryology, Semmelweis University Medical School; and Neuroendocrine Section of the Hungarian Academy of Sciences, Semmelweis Medical University Joint Research Organization, Budapest, Hungary

Received August 25, 1992

Summary. The pineal complex in the mink (Mustela The forerunner of vertebrates is believed to have vison) consists of a larger ventral and a smaller dorsal been four-eyed: two eyes looking laterally as locators pineal. Both organs contain pinealocytes, neurons, glial for perceiving the reflected light from objects in the cells, nerve fibers and synapses in an organization environment, and two eyes looking toward the sky characteristic of nervous tissue. The cellular elements as primitive pineal organs to perceive modifications are arranged circularly around strait lumina. These lumina correspond to the photoreceptor spaces of sub- of solar radiation (see Fig. 10, in VIGH and VIGH- mammalian pineals. A 9+0-type cilium marks the recep- TEICHMANN, 1988). Accordingly, two organs form a tory pole of the pinealocytes which may form an inner- pineal complex in some cyclostomes, teleosts, anur- segment-like dendrite terminal in the pineal lumina. ans and reptiles, while in other vertebrates they are The cilia correspond to outer segments which form photo- represented by a single organ or two anlages which receptor membrane multiplications in the pineal of sub- unite during ontogeny, as do the human pineal (MOL- mammalians and in certain insectivorous and mustelid LER, 1974). Several mammals have both deep and mammals (bat, hedgehog, ferret). Axonal processes of superficial parts of the pineal. The incidence of two the pinealocytes contain synaptic ribbons and terminate pineals lying in parallel has not yet been registered in on intrapineal neurons of both organs. This pattern adult mammals. represents a neural efferentation of the pineal nervous The mink (Mustela vison) is a member of the tissue The axonal processes of pinealocytes also form neurohormonal endings which pierce the perivascular mustelids, which are ancient carnivorous animals. limiting glial membrane in the ventral as well as in the Our examination of the mink aimed to gain further dorsal pineal. The upper pineal ("epipineal") of the mink data on the organization of the pineal in this mam- may correspond to the parapineal, frontal, or parietal malian group as it differs from those generally stud- organs of submammalian pineal complexes. ied. Recently, we have found outer-segment-like cilia Both pineals are encapsulated by the meningeal tissue of photoreceptor cells in the pineal organ of the of the brain stem. Afferent vasomotor axons of the ferret, another mustelid (VIGH and VIGH-TEICHMANN, meninges innervate smooth muscle cells of pineal arter- 1991b). It is already known that the pineal of the ioles. There are corpora arenacea in the pineal arach- mink plays a role in the seasonal timing of gonadal noid and in the pineal nervous tissue, primarily in the activity, moulting periods, pelage composition, etc. ventral pineal. The localization of calcium ions detected around the membrane of pineal cells by pyroantimonate (MARTINET and ALLAIN, 1985). The structure of the cytochemistry suggests membrane activity as the source pineal was investigated by WEMAN and NOBIN (1979), of the calcium ions. The accumulation of calcium by the ROUVET (1982), and TILLET and coworkers (1989). pinealocytes may be due to their neurosensory character. The localization of biogenic amines and melatonin The mink is the first animal described to have both intra- was described by these authors, and the fine structure pineal and meningeal concrements like the human pineal. of the mink pineal was found to be similar to that of

This investigation was supported by the Hungarian OTKA grant No. 1109.

477 478 B. VIGH and I. VIGH-TEICHMANN:

the ferret except that intrapineal neurons were not desmosomes. The upper pineal is in close contact detected in the mink. with the choroid plexus of the suprapineal recess. Our interest in studying the mink was strengthened Both organs contain a small lumen in the center of when we found a double pineal, with both sections the follicular tissue units (Fig. la, c-e). These are containing neuronal perikarya. There were also cal- more prominent in young animals than in adults. cified concretions and meningeal axons innervating Bundles of axons run in these microlumina formed by pineal arterioles in this mustelid. ependymal cells bearing microvilli and a kinocilium and connected by cell binding structures. Dendritic processes of pinealocytes also border these lumina MATERIALS AND METHODS and often form inner segment-like thickenings (Fig. id, e) which contain microtubules, basal bodies and Twenty-seven minks (Mustela visors) aged from new- striated ciliary rootlet fibers. Solitary 9 + 0-type (Fig. born to one year old (sexually matured) and one id, inset) cilia emerge from pinealocytes into the micro- and-a-half year olds of both sexes were received from lumina and/or the intercellular space. The cytoplasm the Maxima stock-farm in Vaskut, Hungary. After of the pinealocytes is characterized by an abundance hexobarbital natrium (AWD Dresden, FRG) narcosis, of ribosomes and mitochondria which are larger than 14 animals were perfused intracardially with 1-4% those of the pineal neurons. An axonal process of the glutaraldehyde; the pineals of the others were im- pinealocytes which contains parallel microtubules mersed in a fixative of 3-5% glutaraldehyde dis- and mitochondria runs to the pineal neurons and/or solved in phosphate or cacodylate buffer (pH 7.2; 300 (their collaterals ?) crosses the limiting glial mem- mosm). The best fixation was obtained with 4% brane (Fig. 2a) to form ribbon-type neurohormonal glutaraldehyde. For the localization and ultrastruc- terminals (Fig. 2a, b) in both the ventral and the tural visualization of calcium ions, the potassium dorsal pineal. Mitoses were often found in the pineals pyroantimonate (PPA) method was used as described of one to four week-old minks. earlier (VIGH et al., 1989b). The material was washed There is a high number of neuronal perikarya in in a buffer, dehydrated in ethanol, and embedded in both pineals. The nerve cells are larger and more Polybed 812 or LR-White (Polyscience, St. Goar, electron lucent than pinealocytes (Fig. 2c), and con- FRG). One micron thick sections were stained with tain small mitochondria, rough surfaced endoplasmic toluidine blue-azure II for light microscopic examina- reticulum and Golgi-areas. Axons presumably emerg- tion. Ultrathin sections stained with uranyl acetate ing from intra-and/or extrapineal neurons form and lead citrate were examined and photographed in synapses on the neuronal perikarya and their den- JEM 6C and OPTON 902 electron microscopes. drites. Some of the axon terminals contain synaptic vesicles and a few granular vesicles; other terminals formed by pinealocytic processes also contain vesi- cle-crowned rodlets (Fig. 2d). A few electron lucent RESULTS profiles (of pinealocytic or neuronal processes ?) contain synaptic spherules (Fig. 2e). Myelinated and Structure of the pineal organs unmyelinated nerve fibers are present intrapineally The mink has a pineal complex located below the and perivascularly. splenium of the corpus callosum. It is composed of a Both pineal organs are covered by a leptomenin- smaller upper, and a larger ventral organ separated geal tissue (Figs. la, b, 3a), the continuation of the from each other by a meningeal layer (Fig. la, b). The corresponding diencephalic meningeal layers. They two pineals are separately connected to the habenu- consist of a stratified arachnoid and the cell layers of lar area of the epithalamus. The dorsal pineal is not the pia mater. Axons of the perivasal nerve bundles a diverticlulum of the ventral one as indicated in (Fig. 3b, c) form synapses on the smooth muscle cells serial sections. Both organs contain pinealocytes, of pineal arterioles (Fig. 3d). glial cells, nerve cells, unmyelinated and myelinated nerve fibers and synapses. The tissue of the pineals is Pineal calcification separated from the pial/perivascular spaces by a basal lamina. Intermediate filament-containing processes There are numerous corpora arenacea in the pineal of glial cells run to the perivascular and outer sur- complex of the mink. They occur predominantly in faces and form a limiting membrane with their end- the ventral pineal organ and its arachnoid; however, feet. The latter are connected by junctional struc- they are absent in the arachnoid of the surrounding tures and are attached to the basal lamina by hemi- brain tissue (Fig. 3a). meningeal acervuli are larger Pineal Organs in the Mink 479

d e Fig. 1 a-e. Light- and electron-microscopic structure of the pineal complex of the mink. a and b. The dorsal (DP) and ventral (VP) pineal separated by a meningeal layer (M). Arrow: Lumen of a follicular tissue unit sectioned through its center. x250. c. Pineal lumen (L) of the ventral pineal of an adult animal. D pinealocytic dendrite, E ependymal cells bearing microvilli and connected by zonulae adherentes. X 18,800. d. Innersegment- like protrusion (IS) bearing a cilium (C) at the end of a pinealocytic dendrite (D) in a photoreceptor lumen (L) of the ventral pineal of a two-week-old animal. E ependymal cells, asterisk ciliary pit. X45,600. Inset: Cross section of a 9+0 cilium. Asterisk ciliary pit. X 51,400. e. Dendritic process (D) of a pinealocyte of an adult animal. E ependymal cells connected to the pinealocytic dendrite by cell-binding structures, IS inner segment-like enlargement, L pineal lumen. X 38,000 480 B. VIGH and I. VIGH-TEICHMANN:

than intrapineal ones: most of the latter are only and glial cells, even in young animals where concre- visible with the electron microscope (Fig. 4a, d). tions are rare or absent. Intracellular deposits are Meningeal concrements may be extremely large in present in some dark cells and their nuclei in older (1 the one-and-a-half-year-old minks. to 1 1/2 year-old) animals. The surface and the layers Using the PPA-method, small Ca-pyroantimonate of the concrements contain variable numbers of precipitates are found in the narrow intercellular space needle-shaped crystals (Fig. 4c). Microconcrements around pinealocytes (Fig. 4b), neurons, nerve fibers often appear inside glial cells (Fig. 4d) and around

a c

b d e Fig. 2 a-c. Fine structure of the pinealocytes, neurons and synapses of the pineal complex. a. Part of a pinealocytic perikaryon (P) and axonal process (X) crossing the line of the glial endfeet (asterisks) and forming a terminal (T) at a pineal vessel (V). X 20,100. b. Synaptic ribbons (arrowheads) containing terminals (T) in the perivascular space. V lumen of a pineal vessel. X 21,200. c. Perikaryon (P) and processes (asterisks) of a nerve cell of the dorsal pineal. N nucleus. X 9,800. d. Axodendritic synapse (arrows) containing synaptic ribbons (R) presynaptically. D dendrite. x 38,000. e. Synaptic spherules (S) in an electron-lucent profile of the ventral pineal. x 32,000 Pineal Organs in the Mink 481

c d Fig. 3 a-d. Details of the pineal meningeal cover. a. Several corpora arenacea (asterisks) in the pineal arachnoid. A arachnoid of the mesencephalic superior colliculus, P pineal tissue. Photomontage. x 420. Inset: Intrapineal acervulus (arrow) in the ventral pineal. x800. b. Nerve bundles (arrows) accompanying pineal arterioles (A). P tissue of the ventral pineal, R red blood corpuscles. X600. c. Detail from the wall of a pineal arteriole. A bundle of axons, L lumen of the vessel, M smooth muscle cells. X 7,800. d. Axon containing synaptic vesicles (V) terminates on the smooth muscle cell (S) of a pineal arteriole. Arrows dense bodies of the smooth muscle cells, F myofilaments. PPA-reaction. x 34,000 482 B. VIGH and I. VIGH-TEICHMANN:

a C

b d

e f Fig. 4 a-f. Calcification in the pineal nervous tissue and pineal arachnoid. a. Acervulus (asterisk) in the pineal nervous tissue of the ventral pineal; note the concentric layers of the concrement. PPA reaction. x9,800. b. Ca-pyroantimonate deposits (black lines) in the intercellular space around pinealocytes. N nucleus of a pinealocyte. PPA reaction. X25,000. c. Needle-shaped crystalls (arrows) inside and around calcified concretions (CA) in the arachnoid of the ventral pineal. x 53,600. d. Small calcified concretions (dark areas) in a glial process / Pineal Organs in the Mink 483 vessels entering the organ. a frontal organ in frogs, and a parietal organ in some The arachnoid is thickened at several places by reptiles (VOLLRATH, 1981). The two components of accumulations of large arachnoid cells. Corpora the pineal complex function differently: in frogs the arenacea usually occur inside these cellular islands. frontal organ contains more cone-type pinealocytes The periacervular cells of the islands contain several while the pineal organ contains more rods (VIGH and mitochondria, Golgi areas and rough-surfaced endo- VIGH-TEICHMANN,1986). In the mink, we have not yet plasmic reticulum. Most of the meningeal corpora found any essential differences in the structural orga- arenacea are surrounded by collagen microfibrils. nization of the two pineals. Regarding form and There are Ca-pyroantimonate precipitates aroud position, the smaller, dorsal pineal ("epipineal") may periacervular cells except at the tight junctions con- correspond to one of the submammalian "accessory" necting arachnoid cells (Fig. 4e). Some periacervular pineal organs, but further data are needed to clarify cells contain various amounts of Ca-pyroantimonate ist exact homology. deposits in their cytoplasm and even in their nuclei In both pineals, the cellular elements are arranged (Fig. 4f). Cells loaded with deposits seem to degener- radially around pineal lumina which are larger in ate and contribute to the formation of acervuli. young animals than in adults. It is known that the photoreceptory tubulo/saccular pineal of cyclostomes and fishes develops into a compact organ with a DISCUSSION follicular structure in higher vertebrates by multiple evaginations of its wall, and that this follicular pineal In contrast with the general view of an exclusively resembles glandular tissues when studied light micro- glandular nature for the mammalian pineal ("pineal scopically (Rio HoRTEGA, 1932; BARGMANN, 1943; gland"), we regard the organ as a complex brain VOLLRATH, 1981). At the electron microscopic level, tissue-"pineal organ"-not only from its develop- however, photoreceptory outer segments containing ment but also from its composition of glial cells, neu- immunoreactive photopigments and extending from rons, synapses and pinealocytes. Cytologically, the pinealocytes into the lumina of the pineal follicles pinealocytes resemble neurosensory cells not only in were demonstrated not only in cyclostomes and fish submammalian vertebrates but also in mammals, start- but also in the pineal of amphibians, reptiles and ing with more primitive ones like insectivores (bat, birds (VIGH-TEICHMANNet al., 1980; VIGH et al., 1982; hedgehog see: VIGH and VIGH-TEICHMANN,1988). This VIGH and VIGH-TEICHMANN,1988; VIGH-TEICHMANN, neurosensory character may explain the ability to con- 1991). Consequently, these follicles have nothing to do centrate calcium ions (see below). It also raises the with glandular acini, but are receptory structures, question of whether the pinealocytes of all mammalian their lumina being analogous with the photoreceptor species have indeed lost their direct photosensitivity space of the retina. Therefore, the appearance of (VIGH and VIGH-TEICHMANN,1989a; VIGH-TEICHMANN follicles during evolution does not indicate a gradual and VIGH, 1992). Like insectivores, the mustelids are transformation of the nervous tissue of the photo- ancient carnivores, representing a phylogenetically receptive pineal into a glandular one, but it repre- old group of mammals. In the mink, the high number sents a way to expand the wall of the organ. The of pineal lumina and pinealocytic axons forming radial arrangement of pineal cells around lumina, as synapses on secondary pineal neurons as well as the found in the mink, are similar structural units; their presence of pinealocytic cilia may be connected to lumina correspond to the photoreceptor spaces of this ancient character. The presence of two pineals as submammalian pineals containing inner segments found in the mink seems to be further evidence in and photoreceptory cilia of pinealocytes (Fig. 5). favour of the similarity of its pineal complex to that In our opinion, the endocrine character of pinealo- of submammalians. cytes (see also: LEONHARDT, 1967; WARTENBERG, As already mentioned, two members of a pineal 1968; PANG et al., 1989) is cytologically represented complex are present in some cyclostomes, teleosts, by their neurohormonal axon terminals (=terminal anurans and reptiles. Besides the pineal organ, there axon enlargement /a/, with accumulations of synap- is a parapineal organ in the lamprey and bony fishes, tic and/or granular vesicles /b/, hemidesmosomes

/, containingd filaments (arrow). PPA reaction. x 18,800. e. Accumulation of a high amount of Ca-pyroantimonate eposits (black lines) in the enlarged intercellular space around arachnoid cells. Encircled area : tight junction devoid of deposits, R rough-surfaced endoplasmic reticulum in a meningeal cell. x 27,300. f. Enlarged intercellular spaces filled with Ca-pyroantimonate deposits close to a meningeal acervulus (A). C periacervular cell free of deposits, asterisk intranuclear deposits of a cell bordering the acervulus. PPA reaction. x 18,600 484 B. VIGH and I. VIGH-TEICHMANN:

Fig. 5. Scheme of the follicular organization of the pineal tissues of the mink: details of the wall of a photoreceptory follicle. A pineal arteriole with smooth muscle cells (M) innervated by meningeal axons (X), C 9+0-type pinealocytic and 9+2-type ependymal cilia in the photoreceptor lumen (asterisk) of a follicle, E ependymal cell, F glial endfeet, G glial cell, H neurohormonal axon terminals of the pinealocyte, L basal lamina, MI intermediate filaments in the glial processes, N pineal neuron, P pinealocytes, R synaptic ribbon containing terminals, S synaptic spherules, SC Schwann-cell, arrows aff erentation and efferentation of the pineal. and/or synaptic bars, spherules /c/, facing the basal mentioned are formed by axons of the parapinealo- lamina /d/). These terminals pierce the superficial/ cytes bearing well developed outer segments. There- perivascular limiting glial membrane of the pineal fore, the appearance of the pineal endocrine struc- nervous tissue (VIGH et al., 1986a). In the mink, tures is not necessarily connected to the reduction of pinealocytes of both organs form neurohormonal outer segments as seen in reptiles, birds or mammals endings on the surface of the pineal tissue. Such (VOLLRATH, 1981, VIGH and VIGH-TEICHMANN, 1988; neurohormonal terminals are needed for the release COLLIN et al., 1989). of hormones at certain locations along the nervous In our opinion, neurohormonal terminals of pineal system (e.g. neurohypophysis, median eminence, uro- organs represent an alternative efferentation besides physis, etc.), because usual nervous tissue is separat- a synaptic one, both being developed to a variable extent ed from the perivascular space by the limiting glial in the animals studied. We do not know whether the membrane present in the pineals of the mink as well. axon collaterals of the same pinealocytes or of two The presence of secretory structures is not restrict- different cells respectively form the synapses and the ed to pinealocytes of higher vertebrates. Neurohor- neurohormonal terminals. monal endings already exist in the lamprey, a cyclo- Contrary to the general view that the mammalian stome, one of the most simple vertebrates having pinealocytes are simple glandular cells, they actually a pineal complex (MEINIEL, 1969, 1971; VIGH, 1987; exhibit structures typical of a photosensory cell by VIGH and VIGH-TEICHMANN, 1988). The structural and their 9+0-type cilia and innersegment-like processes electrophysiologycal investigation of the lamprey (WOLFS, 1965; CLABOUGH, 1971; ZIMMERMANN and pineal confirm its photosensory character (MoRITA Tso, 1975; MOLLER, 1986), by their synaptic ribbons et al., 1989, 1991). The neuroendocrine terminals found in about 30 mammalian species (MCNULTY and Pineal Organs in the Mink 485

Fox, 1922) and by their axonal processes forming pineal nerve cells in both the dorsal and ventral synapses on secondary pineal neurons in some species pineals of the mink. Intrapineal neurons are thought (VIGH and VIGH-TEICHMANN,1975; VIGH et al., 1975, to be peripheral autonomic ganglion cells (KENNY, 1986b; VIGH-TEICHMANN, 1991), and further by the 1961; KAPPERS, 1965 ; ROMIJN,1975 and others). In the presence of substances known for their role in retinal mink, however, they differ from peripheral auto- phototransduction (KALSOW and WACKER; 1977; nomic neurons (see also : DAVID and HERBERT, 1973, VIGH-TEICHMANNet al., 1986; ABE and SHINOHARA, in the ferret; ICHIMURAat al., 1986; ICHIMURA,1992, 1990; PALCZEWSKIet al., 1990; VIGH-TEICHMANNand in the monkey), by the absence of satellite cells and VIGH, 1992; LOLLEY et al., 1992). basal lamina, i.e., they are located in the pineal nerv- The membrane of the ciliary outer segments does ous tissue proper like other nerve cells of the central not form lamellar multiplications in most of the mam- nervous system. The pineal nerve cells of the mink mals studied but remains a smooth 9+0 cilium extend- receive some axon terminals of unknown origin, ing into the reduced lumina of the photoreceptory while others are formed by synaptic ribbon-con- follicles mentioned and/or into the intercellular space. taining endings of pinealocytec axons as described in In some mammals like the bat, hedgehog, rabbit and the pineal of the ferret, rat, cat, hedgehog, guinea pig ferret, however, the cilia of the pinealocytes may and monkey (DAVID and HERBERT, 1973; VIGH et al., form bulb-like enlargements and/or contain vesicles 1975; ICHIMURAet al., 1986; VIGH-TEICHMANN, 1991; like developing retinal outer segment (PEvET et al., VIGH-TEICHMANNet al., 1991; VIGH-TEICHMANN and 1977; VIGH and VIGH-TEICHMANN, 1981, 1988, 1991b; VIGH, 1992). VIGH et al., 1986b; VIGH-TEICHMANNand VIGH,1986). Some of the intrapineal neurons may be interneu- These structures may be responsible for the non- rons (VIGH-TEICHMANNet al., 1991) while the axons retinal photic modulation of melatonin's pineal mech- of others seem to leave the pineal tissue. Intrapineal anisms in mammals as demonstrated by QUAY (1989). neurons project to the habenular, pretectal and other In contrast, most physiologists deny that the mam- brain stem nuclei in submammalians. The two com- malian pineal is sensitive to direct light; it is gener- ponents of these pineal complexes have different con- ally accepted in the literature that light information nections (cyclostomes: PUZDROWSKIand NORTHCUTT, reaches the pineal via autonomic nerves. Neuro- 1989; fishes: EKSTROM, 1984; HAFEEZ and ZERIHUN, anatomically, however, a retino-pineal connection 1974; BHARGAVE,1973; amphibians: MAUTNER,1965: would be more evident via a direct afferent projection PAUL et al., 1971; PAUL, 1972; ZIMMERMANNand PAUL, from retina-related centers of the brain stem (VIGH- 1972; reptiles: TROST, 1954; KUMMER-TROST, 1956; TEICHMANN,1991) as peripheral nerves usually do not ORTMAN,1960; KAPPERS, 1976). In the human pineal, reenter the central nervous system to "innervate" the we found axons of Intrapineal neurons running to the nervous tissue proper of the brain. posterior commissure (VIGH and VIGH-TEICHMANN, In both pineals of the mink, the receptory pole of 1991a), and in the cat to both the posterior and the pinealocytes is marked by an inner-segment-like habenular commissures (VIGH and VIGH-TEICHMANN, dendritic process protruding into the pineal lumina 1991a; VIGH-TEICHMANNet al., 1991; VIGH-TEICHMANN and by a 9+0-type cilium; these are homologous to and VIGH, 1992). Since these bundles mainly represent the ciliary outer-segment of the submammalian pinealo- connections with neighbouring brain stem areas, and cytes. Ontogenetically, young pinealocytes, presum- the connections of the epithalamus are rather conser- ably derived from mitoses present in one to four vative during evolution (CLARA, 1959), we think that week-old minks, do not have direct contact with the some of the intrapineal neurons in mammals may lumina since their cilia extend into the intercellular project to the same brain stem nuclei as do the sub- space (Fig. 5). mammalian pineal neurons. S-antigen immunoreac- We could not find membrane multiplications on the tive mammalian pinealocytes were described as pro- cilia in any of the minks studied. The lack of ciliary jecting directly into habenular and pretectal regions membrane disks may result in a lower sensibility of by KORF and his coworkers (1986). In the two com- the mammalian pinealocytes when compared to those ponents of the mink pineals the connections may of submammalians. We have to take into considera- possibly be different, though they are not yet known. tion that the size of the pineal increases during Therefore, further studies are necessary to find the development by sac-like f oldings of its wall, and that site of termination of the axons of secondary pineal the multiplication of the individual ciliary membrane neurons. may be replaced by the high number of pinealocytes The "capsule" of the pineal organs of the mink is a in phylogenetically more developed animals. portion of the meninges of the diencephalon and As in the ferret, there is a large number of intra- corresponds to the arachnoid and pia mater of the 486 B. VIGH and I. VIGH-TEICHMANN: leptomeninx. In these meninges, there are unmye- finding corroborated by HUMBERT and PEvET (1991). linated axons that innervate smooth muscle cells of In the present work, fine Ca-pyroantimonate deposits pineal arterioles. Though several works have con- were found on the cell membrane around pineal cells sidered the autonomic afferentation of the mam- in young animals where acervuli were rare or absent. malian pineal (MOLLER, 1992), and experimental In accord with the opinion of KRSTIC (1985), we studies have reported on important effects of these explain both this phenomenon and the formation of nerves on pineal function little is known about their acervuli as arising from the membrane activity of vasomotor role. In earlier studies we proposed pineal cells which are able to expel calcium ions. The vasomotor and viscerosensory functions for the nerve accumulation of a higher number of pinealocytes fibers of the pineal meninges (VIGH-TEICHMANNet al., and/or the higher membrane activity in mammals 1973; VIGH and VIGH-TEICHMANN, 1988, 1989a; VIGH- may expain the frequent formation of acervuli in TEICHMANN,1991 ; VIGH-TEICHMANNand VIGH, 1992). these animals when compared to submammalians. For further clarification of the morphological side of The high calcium exchange of pinealocytes may be this problem, studies to identify the origin of these connected to their photosensory nature, since outer meningeal axons are already in progress. segments of pinealocytes of submammalians and Our earlier investigations demonstrated an accu- retinal photoreceptors contain a considerable amount mulation of calcified concretions in the pineal arach- of calcium (VIGH-TEICHMANNand VIGH, 1992). Cal- noid of the rat and bat as well as in some birds (VIGH, cium ions are known to cross the cell membrane 1987; VIGH and VIGH-TEICHMANN, 1988, 1989a, b; during photoreceptoon, e.g., in a dark-adapted" state VIGH et al., 1989a, b; VIGH-TEICHMANN and VIGH, they are concentrated in the intracellular side of the 1992). In human pineals, we have found a similarity photoreceptor lamellae of retinal outer segments, between intrapineal and habenular acervuli. Most of while in light-adapted outer segments, calcium ions the calcium ions around concrements of the human occur on the intracellular, cisternal side of the mem- and rat pineal arachnoid appear extracellularly when branes (ATHANASSIOUSet al., 1984; VIGH and VIGH- studied by pyroantimonate cytochemistry (VIGH,1987; TEICHMANN, 1989a; VIGH-TEICHMANNand VIGH, 1992). VIGH and VIGH-TEICHMANN,1989a ; VIGH-TEICHMANN In the mink, one important step of calcification and VIGH, 1992). With the exception of some birds, may be represented by the accumulation of calcium the presence of corpora arenacea in the pineal organ ions in the enlarged intercellular spaces as well as is usually restricted to mammals including man intracellularly in some pineal and miningeal cells. (KRSTIC and GOLAZ, 1972; LUKASZYK and REITER, The barrier effect of the tight junctions found among 1975; KRSTIC, 1986; WELSH, 1984, 1985). Intrapineal arachnoid cells may facilitate the formation of concrements are numerous in the Mongolian gerbil maningeal concrements as suggested by the absence (JAPHA et al., 1976; WELSH and REITER, 1978; KRSTIC, of Ca-pyroantimonate deposits at tight junctions and 1985; KARASEKand REITER, 1992) which serves as the the larger size of meningeal acervuli when compared main experimental animal for studying pineal calci- to intrapineal ones in the mink. fication. An intercellular accumulation of Ca-pyroanti- In the present work, we found corpora arenacea in monate deposits around intrapineal corpora arenacea the pineal nervous tissue proper and in the perivas- was found in the Mongolian gerbil by KRSTIC (1985), cular spaces and in the arachnoid of both pineals of and by us around meningeal concrements in rat and the mink. They are predominantly present in the human pineals (VIGH et al., 1989; VIGH and VIGH- arachnoid of the ventral pineal but absent in the TEICHMANN, 1989a), while WELSH (1984, 1985) and arachnoid of the neighbouring brain areas. Meningeal KRSTIC (1986) described the presence of cations inside calcification was found in other brain areas as well pinealocytes of the gerbil. We found concrements (VIGH et al., 1989b), but not to the same extent as in inside glial cells in the mink. WELSH (1984, 1985) and the pineal meninges. Therefore, we think that the WELSH and REITER (1978) believe the pinealocytec meningeal calcification is related to the intrapineal vacuoles in the gerbil to contain the initial crystal- one. On the other hand, pia mater cells follow vessels lization nuclei of the concrements. penetrating the organ and may contribute to the The concentric layers of the acervuli contain formation of intrapineal concrements as well (Rio- different amounts of calcium (MABIE and WALLACE, HORTEGA, 1932; ARIETI, 1954; SCHARENBERG and 1974). We have found a correlation between the num- Liss, 1965; VIGH, 1987). ber of layers of the largest acervuli and the corre- Using the Kossa method, we detected a higher sponding age of human pineals (VIGH-TEICHMANN calcium concentration in the pineal than in the sur- and VIGH, 1992). This suggests that the formation rounding brain tissue in the rat (VIGH et al., 1989b), a of these layers is connected to circannual changes in Pineal Organs in the Mink 487 the calcium level of the organ and due to its special ICHIMURA, T., T. ARIKUNI and P. H. 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