Cell Tiss. Res. 157, 165--184 (1975) 9 by Springer-Verlag 1975

Uhrastructure of Pars nervosa and Pars intermedia of the Lamprey, Lampetra tridentata* K a z u h i k o Tsuneki Misaki Marine Biological Station, University of Tokyo, Miura, Kanagawa, Japan Aubrey Gorbman Department of Zoology, University of Washington, Seattle, Washington, U.S.A. Received November 1, 1974

Summary. The pars intermedia of the adult lamprey is separated by perivascular spaces and a capillary plexus from the pars nervosa. No penetration of nerve fibers into the pars intermedia was found. The pars nervosa, which constitutes the posterior wall of the infundibulum, consists of an ependymal layer and a fuchsinophilic fiber layer; the latter contains at least four different types of axonal endings. The pars intermedia is avascular and is composed of a small proportion of non-secretory cells and a large proportion of secretory cells. The secretory granules in the cells of the pars intermedia seem to be discharged toward the capillaries that separate the pars intermedia from the pars nervosa. Although no direct nervous or vascular connections were found between the pars nervosa and pars intermedia, a mechanism of control of secretory activity in the pars intermedia cells by the central nervous system appears likely. Key words: Pituitary gland, posterior - - Hypothalamo-hypophyseal system - - Cyclostomes - - Electron microscopy.

Introduction The secretory a c t i v i t y of t h e cells of t h e p a r s i n t e r m e d i a of v e r t e b r a t e s is generally responsive to changes in b a c k g r o u n d a n d i n c i d e n t a l e n v i r o n m e n t a l light, a n d t h u s i t is regulated, a t least in p a r t , b y t h e central nervous system. This r e g u l a t i o n has been a t t r i b u t e d to secretion of p e p t i d e r g i c h y p o t h a l a m i c neuroh o r m o n e s (MSH-releasing h o r m o n e a n d / o r MSH-release inhibiting hormone, Schally et al., 1973), p r e s u m a b l y delivered to t h e p a r s i n t e r m e d i a t h r o u g h t h e blood. A c t i v i t y of t h e p a r s i n t e r m e d i a also has been t h o u g h t to be r e g u l a t e d t h r o u g h m o n o a m i n e s (Knowles, 1965; E n e m a r et al., 1967 ; I t u r r i z a , 1969 ; Meurling et al., 1969; Meurling a n d BjSrklund, 1970; Wilson a n d D o d d , 1973). The p o s s i b i l i t y t h a t o c t a p e p t i d e n e u r o s e c r e t o r y m a t e r i a l s are also r e g u l a t o r s of t h e p a r s i n t e r m e d i a can n o t be ruled o u t (Etkin, 1962; Mellinger, 1963; Knowles, 1965 ; P a n d a l a i a n d Sheela, 1969). S y n a p t o i d j u n c t i o n s between p e p t i d e r g i c a n d / o r m o n o a m i n e r g i c n e u r o s e c r e t o r y axons a n d intrinsic cells of p a r s i n t e r m e d i a are f o u n d in various groups of t h e v e r t e b r a t e s ( B a r g m a n n et al., 1967; N a k a i a n d

Send oHprint requests to: Kazuhiko Tsuneki, Misaki Marine Biological Station, University of Tokyo, Miura, Kanagawa, Japan 238-02. Aubrey Gorbman, Department of Zoology, University of Washington, Seattle, Washington 98195, U.S.A. * This study was aided by a grant from the U.S. National Science Foundation, Grant @ GB 27486.

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Gorbman, 1969; Z a m b r a n o , 1970; Chevins, 1972, etc.). W h e n found, such synaptold relationships have been t a k e n as indication of direct neurogenic control of pars i n t e r m e d i a cells (see also Olsson a n d G o r b m a n , 1973). On the contrary, such i n n e r v a t i o n has n o t been seen in the sturgeon (Polenov et al., 1972) or in the pars i n t e r m e d i a of certain reptiles (Rodrlguez a n d La Pointe, 1970; W e a t h e r h e a d , 1971). The l a m p r e y is the most primitive v e r t e b r a t e with a distinct pars intermedia. A n MSH-like hormone is extractable from the p i t u i t a r y gland of lampreys (Lanzing, 1954) a n d its role i n changes in body color has been assessed (Larsen, 1965). F u r t h e r m o r e , it has been suggested t h a t external light stimuli m a y affect the a c t i v i t y of pars intermedia cells t h r o u g h central nervous relationships even in the lamprey, at least in m e t a m o r p h o s e d Mordacia mordax (Eddy a n d Strahan, 1968) a n d in a d u l t Lampetra planeri (Joss, 1973). Direct nervous innerr a t i o n has been considered as a possible mode of secretory control b y the b r a i n of the lamprey's pars intermedia, because blood capillaries are a b s e n t from the pars i n t e r m e d i a itself (Gorbman, 1965), a n d because contact of some nerve fibers with the intrinsic cells of pars i n t e r m e d i a was observed in ammocoetes of Lampetra planeri (Miiller, 1965). I n order to examine more closely the question of functional control of the l a m p r e y ' s pars i n t c r m e d i a we elected to s t u d y the fine structure of the pars n e r v o s a - - p a r s i n t e r m e d i a j u n c t i o n as well as the p a r e n c h y m a of the pars intermedia itself. Studies of the u l t r a s t r u c t u r e of the pars nervosa a n d pars i n t e r m e d i a of lampreys have been few a n d brief (Miiller, 1964, 1965; Rodriguez, 1971; Larsen a n d Rothwell, 1972). The u l t i m a t e value of such a s t u d y a n d the i n t e r p r e t a t i o n s derived from it is in the basis t h a t it forms for a n y speculations concerning the evolution of secretory control b y the b r a i n of the f u n c t i o n of pars intermedia. A n obvious goal of the s t u d y has been the detailed delineation of a n y possible nervous and/or vascular relations between the pars i n t e r m e d i a a n d the a d j a c e n t pars nervosa.

Material and Methods Adult lampreys of both sexes, Lampetra tridentata (Richardson), were collected from the Columbia river at the Bonneville Dam in May in the course of their upstream migration. They were in aerated 30 gallon covered plastic tanks at a regulated temperature of 10~ until sacrificed. The total lengths of the specimens varied from 53 to 72 cm. The brains of five lampreys were fixed in Bouin's solution, and serial Paraplast 6 it sections were stained with Gomori's paraldehyde fuchsin (AF) and counterstained in order to observe the general organization of the hypothalamo-hypophysial system. For electron microscopy, the hypothalamohypophysial regions of four fish were fixed in double aldehyde fixative for 3 to 4 hours at room temperature (Karnovsky, 1965). The final pH of the fixative was 7.2. After washing with ice-cold phosphate buffer, the tissues were postfixed in 1% osmium tetroxide buffered with s-collidine (final pH 7.0). The pituitary regions of two lampreys were fixed in phosphate buffered (pH 7.2) 1% osmium tetroxide alone for 150 minutes under ice-cold conditions (Millonig, 1962). The tissues were embedded in Epon 812 through a graded series of ethanol and propylene oxide. They were cut by use of a glass knife with the Porter-Blum ultramicrotome. Thick sections for the orientation of tissues were stained with toluidine blue in borax solution. The thin sections mounted on uncoated copper grids were stained with uranyl acetate (Watson, 1958) and lead citrate (Venable and Coggeshall, 1965). The photographs were taken with either the JEOL 100B or RCA EMU-3G electron microscope.

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Fig. 1. Cross section of the anterior part of the pars nervosa--pars intermedia complex of Lampetra tridentata. No prominent accumulation of fuchsinophilic material can be seen in this region of pars n~rvosa (PN). Capillaries are usually absent at this level of the boundary between pars nervosa and pars intermedia (PI). Note the fuchsinophilic cell processes of the intermedial cells abutting on the pars nervosa. Enlarged view of a section of the part enclosed by the r~ctangle is shown in Fig. 3. CP capillary, CT connective tissue, FS shallow fibrous septum, I I I third ventricle. AF staining. • 250 Fig. 2. Parasagittal section of the pars nervosa (PN)--pars intermedia (PI) complex. The boundary of these two tissues is accentuated by dotted lines. Notice the distinction between the ependymal layer above and the fuchsinophilic nerve fiber layer in the pars nervosa. Some fuchsinophilic fibers seem to penetrate the surface of the pars intermedia (arrows). Other abbreviations are the same as those in Fig. 1. AF staining. • 400

Observations General Organization o[ the Pars nervosa - - Pars intermedia Region Some cells in the preoptic nucleus seem to be bipolar, one process following the preoptico-neurohypophysial tract, the other e x t e n d i n g toward the t h i r d ventricle. The m a i n preoptico-neurophysial neurosecretory tracts proceed toward the v e n t r a l side of the h y p o t h a l a m u s a n d enter the pars nervosa at its anterior border. The pars nervosa is a simple sac forming the posterior wall of the i n f u n d i b u l u m . I t consists of a j u x t a - v e n t r i c u l a r e p e n d y m a l layer a n d a fuchsinophilic nerve fiber layer which is apposed to the pars i n t e r m e d i a b u t separated from it b y a t h i n layer of connective tissue which contains a blood capillary plexus. The layer of connective tissue is occasionally relatively thicker at some places in the posterior p a r t of the pars n e r v o s a - - p a r s i n t e r m e d i a complex. Blood capillaries are f r e q u e n t l y a b s e n t a t the very anterior b o u n d a r y region between pars nervosa a n d pars i n t e r m e d i a (Fig. 1) as well as in the postero-dorsal region. The connective tissue layer does n o t seem to be p e n e t r a t e d by nerve fibers of the pars nervosa. Straight fnchsinophilic fibers occasionally seem to i n v a d e the pars i n t e r m e d i a ; however, their structure is t h a t of bundles of collagen fibers; their appearance is different from the g r a n u l a r neurosecretory fibers (Fig. 2). The fact t h a t these fibrous

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connections between pars nervosa and pars intermedia are mainly observed in parasagittally sectioned preparations, which frequently pass tangentially between the nervous and glandular tissue, supports the conclusion that they are collagenous structures. The pars intermedia is composed primarily of m a n y glandular cells. These stain with AF to various degrees. The thickness of the pars interinedia usually corresponds to about 20 cells. The uppermost dorsal zone of the pars intermedia consists principally of intensely fuchsinophilic cell processes of secretory cells which abut on the capillary walls mentioned above and on shallow connective tissue septa in the pars intermedia; such septa occur only infrequently (Fig. 1). The fuchsinophilia is generally relatively weak in the cells in the middle portion of pars intermedia. Some strongly fuchsinophilic cells appear at the ventral surface of the pars intermedia. In this region, blood capillaries enclose the pars intermedia incompletely. The parenchyma contains no blood capillaries; it is vascularized only on the surface, as shown earlier by ink injection in the adults of Petromyzon marinus (Gorbman, 1965).

Ultrastructure o/the Pars nervosa, in Detail The pars nervosa is divisible into two layers: ependymal and nerve-fiber layers (Fig. 3). The cpendymal layer usually consists of a single layer of ependymal cells. Microvilli and cilia are common at the apical surfaces of ependymal cells (Fig. 4). The number of microvilli varies considerably among ependymal cells. Coated vesicles and pinocytotic vesicles are often found in this region (Fig. 5). The concave surface of pinocytotic vesicles is coated with dense material. In addition, the apical cytoplasm contains m a n y vacuoles and vesicles of various sizes, lamellatcd bodies of varying density and shape, possible glycogen granules as well as ordinary cell organelles. Prominent large colloid droplets with diameters of 1.5 to 2 Ez are also usually encountered, some of which seem to discharge their contents into the infundibular recess. The surface of the colloid droplets is adorned with tiny dense particles. The droplets are round in shape in the material prefixed with aldehydes, however, they have an indented outline in preparations made without prefixation. Peculiar associations between colloid droplets and lamellated vacuoles, or between elongated crescent-shaped mitochondria and vacuoles are frequently observed (Fig. 5). The space between ependymal cells is sometimes considerably widened and contains m a n y mierovilli (Fig. 4). Numerous pinocytotic vesicles and coated vesicles are also encountered in the cytoplasm facing the spaces (Fig. 4, inset). Such intercellular spaces are open apically to the third ventricle but they are generally closed at the nuclear region by a junctional apparatus (Fig. 4). The boundary between ependymal cells is sometimes complexly folded. Processes of ependymal cells extend completely through the pars nervosa to the pericapillary space between pars nervosa and pars intermedia and a b u t on it. The nerve-fiber containing region is roughly divisible into hilar and palisade layers, there being a relatively greater abundance of ncurosecretory axons and endings in the latter ; however, the distinction between them is not sharp especially in the anterior part of the pars nervosa. In the nerve-fiber region, there are axons containing neurosecretory granules as well as non-neurosecrctory axons (Fig. 6). The latter are most frequent in the central hilar region, being usually smaller in

Fig. 3. Anterior region of the pars nervosa (see locus outlined in Fig. 1). The ventricular surface (top) of the pars nervosa is lined b y ependymal cells (EC). In the fiber layer, there are neurosecretory fibers (NF) and ependymal processes (EP) in which m a n y colloid droplets (CD) a n d vaeuolated formations (VF) are a b u n d a n t . Notice the ramified incursions of the basal lamina of the pericapillary space (PCS) into the pars nervosa (at arrow for example). The capillaries (CP) only partly separate the pars intermedia (PI) from the pars nervosa in this region. CL cilia, ED endothelium, H B Herring body, P T pituicyte or subependymal cell, I I I third ventricle. OsO 4 fixation, x 3 500

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Fig. 4. Ependymal cells of the pars nervosa. There are m a n y microvilli (MV) at the apical surface and in the space between adjacent ependymal cells. Notice the pinocytotic vesicles on the cell membrane facing the intercellular space (arrows in the inset). CL cilia, D B dense body, GA Golgi apparatus, J A junctional apparatus, M mitochondria, N nucleus of ependymal cell, I I I third ventricle. O s Q fixation, x 12500 (inset, • 10900) Fig. 5 The ventricular surface of ependymal cells. Note the peculiar association between vacuolar formations (VF) and colloid droplets (CD) or elongated mitochondria (M). CU cell of unknown nature, C V coated vesicle, M V microvilli, P V pinocytotic vesicle. 111 third ventricle. Os04 fixation, x 13300

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Fig. 6. Parenchymal region of the pars nervosa. Arrow indicates the association between a colloid droplet (CD) and a vacuolar formation (VF). There are many vesicular and tubular structures in the ependymal process (EP). D B dense body, N nucleus of a pituicyte or subependymal cell, N N F non-neurosecretory fiber, I type I axon, I I I type I I I axon. I V type IV axon. OsO 4 fixation, x 13100 Fig. 7. Neurosecretory axons in the pars nervosa fixed with osmium tetroxide alone. Notice the axons (arrows) surrounded by the ependymal process. DD dense droplet, M B myelinated body containing neurosecretory granules, I I I type I I I axon. x 11600 Fig. 8. Neurosecretory axons in the pars nervosa prefixed with aldehydes. Note the irregularly shaped dense granules in the type VI axons (VI). I I type I I axon, I I I type I I I axon. x 14800

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diameter and including m a n y neurotubules. A few neurosecretory granules occasionally appear in the axons of small diameter. The ependymal processes are characterized b y fine ependymal filaments and tubules, which generally run parallel to the membrane wall of the process. Mitochondria, dense bodies and large colloid droplets are also c o m m o n components in the ependymal process. The colloid droplets frequently associate with the vacuolated membrane system in the process as well as in the perinuclear region (Fig. 6). Many glycogen granules and vesicular or tubular formations of varying size and shape are also commonly found in the ependymal process. Subependymal cells and pituicytes are observed in a limited n u m b e r in the pars nervosa. T h e y generally have a denser cytoplasm t h a n t h a t of ependymal cells, and numerous glycogen-like granules. Vesicular structures and dense lamellated bodies are also scattered in the cytoplasm of the pituicyte. I n the palisade region, six to eight kinds of axons or axonal endings could be observed (Figs. 7-9). The endings of the t y p e - I axon contain only small " e m p t y " vesicles with diameters of 440 to 480 A (Figs. 6, 9). Since the n u m b e r of these fibers is small, the possibility t h a t this is a regionally limited class of axons t h a t contains additional granules or vesicles in other sections cannot be excluded. T y p e - I I axons, which contain relatively small granules with diameters of 700 to 800 A as well as small vesicles also are only rarely found (Figs. 8, 9). T y p e - I I ' axons differ from the t y p e I I only in t h a t t h e y contain lucent vesicles with diameters 750 to 900 A instead of dense granules (Fig. 9). The population of the t y p e - I I ' axons is comparatively large compared to t h a t of the type I I in the material without prefixation. The occurrence of this type of axons is very infrequent in the material prefixed with aldehydes. T y p e - I I I axons and terminals are the most a b u n d a n t (Figs. 6-9). T h e y have large electron dense granules from 1600 to 2000 A in diameter and small e m p t y vesicles which together with mitochondria, fill the section plane. The electron density of the granules varies even within one axon. These granules are spherical in shape in the material simply fixed with osmium tetroxide, however, prefixation confers on t h e m a slightly polygonal outline and makes the content less dense (compare Fig. 7 with Fig. 8). Large round, dense droplets with diameters of 6000 to 8000 A are occasionally encountered in the t y p e - I I I axons (Fig. 7). T y p e - I I I ' nerve endings contain electron-lucent vesicles with diameters of 1400 to 2200 A and small e m p t y

Fig. 9. Palisade region of the pars nervosa. Many neurosecretory axon endings and ependymal end feet (EP) terminate on the basal lamina (BL). Notice that no nerve fibers penetrate the basal lamina or traverse the pericapillary space (PCS), containing collagen fibrils. A triangle (upper right) indicates a possible synaptoid contact between type I axon (?) and ependymal process. Short arrows show pinocytosis in the endothelium (ED). Long arrows indicate coated vesicles in the ependymal end feet. CP capillary lumen, I, II, II', III, III" and I V represent the types of axons so designated. OsO4 fixation, x 13500 Fig. 10. A non-granulated cell (.NGC) in the region of the pars intermedia facing the basal lamina (BL). Arrows indicate slender cytoplasmic processes of non-granulated cells. DB dense body, M mitochondria, PCS pericapillary space, SG secretory granules in the cytoplasmic pole of the pars intermedia cells. OsO4 fixation, x 10900 Fig. 11. Processes of non-granulated cells are connected by desmosomes. PCS pericapillary space, SG secretory granules in pars intermedia cells. Karnovsky's fixation, x 11500

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vesicles (Fig. 9). Type-IV axons, which contain dense granules from 1000 to 1300 A in diameter, together with small vesicles, are few in number and only are found in material without prefixation (Fig. 6). Type-V and type-VI fibers, which can be distinguished from each other according to the size of granules contained, are only observed in the pars nervosa prefixed with aldehydes (Fig. 8). Both kinds of axons contain granules t h a t are characteristically irregular in shape. Some are round or oval, others are reminiscent in outline of a tailed comet. These granules usually have a distinct limiting membrane. In type-V axons, which are few in number, the granules range between 1000 to 1400/~ in shortest diameter. The type-VI fibers are more abundant than the type V, but fewer than the type I I I . They contain granules from 1500 to 2000 A in the short axis, as well as mitochondria. Type-V and typc-VI axons have small e m p t y vesicles. Their granules have a high electron density. These two kinds of axons occur mainly in the central region of the pars nervosa. The axonal types that are seen only in the material with prefixation obviously m a y correspond to some of the types observed in material without prefixation. Granule types that are typically found in separate nerve axons, like types I I I and I I I ' , are occasionally encountered in the same axon. In an extreme case, the granules generally restricted separately to II'-, I I I - and I I I ' - t y p e axons are observed together in one axon terminal. Such mixtures of granules and vesicles are mainly found in the ventral part of the pars nervosa. Tiny dense particles, regarded as glycogen vesicles, are found in various densities in all types of axons. They generally are absent from the pars nervosa prefixed with aldehydes. Large myelinated bodies containing electron dense granules are occasionally observed (Fig. 7). Small dense lamellated bodies are frequently found in Herring bodies which occur sometimes just beneath the ependymal layer (Fig. 3). The granules filling the dilated axoplasm of the Herring body vary in density, some of them being relatively dense, others lucent. Ultrastructural Features o/the Relationship between the Pars nervosa and Pars intermedia These two tissues are separated from each other by perivascular or intervascular spaces. ~qo interruptions of the perivascular space or penetrations of nerve fibers across the fibrovascular septum into the pars intermedia were encountered in the regions from the anterior part, middle region, to the posterior part of the pars nervosa--pars intermedia complex. The width of the tissue wall between pars nervosa and pars intermedia generally varies from 2 to 20 ~z. The perivascular space facing the pars nervosa consists of a basal lamina 600 to ] 000 A in width and layers of collagen fibrils embedded in amorphous substrate (Fig. 9). The bundles of collagen fibrils run in various directions. Short extensions and ramifications of the basal lamina into the palisade layer are common. Neurosecretory nerve endings are usually directly contiguous with the basal lamina, although the interposition of ependymal or glial end feet was also observed (Fig. 9). Tubular or vesicular formations, coated vesicles and irregular dense bodies are occasionally found in these end feet. Exocytosis of neurosecretory granules into the perivascular space was not observed.

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Fenestration of the endothelial cells of the capillary walls was not observed; however, small vesicles are commonly found in the endothelial cytoplasm, some of which represent pinocytosis, presumably both from the connective tissue and the capillary lumen (Fig. 9). In addition, the endothelial cytoplasm contains mitochondria, dense lamellated bodies, colloid droplets, rough-surfaced endoplasmic reticulum and small tubular formations. In the nucleus of the endothelial cell there is peripheral accumulation of chromatin material. Each endothelial cell is joined to adjacent cells by well developed foldings, and sometimes by junctional complexes. The pericapillary space contacting the pars intermedia is similar in structure to that facing the pars nervosa (Fig. 10). Although the cytoplasmic processes of many pars intermedia cells directly contact the basal lamina of pars intermedia, the interposition of cytoplasmic elements of non-granular cells between the basal lamina and pars intermedia cells occurs frequently (Fig. 10). The cytoplasmic processes of these agranular cells characteristically contain elongated mitochondria and fine filaments. They are sometimes connected with desmosomes to each other (Fig. 11). The cytoplasmic poles of pars intermedia cells that contact such agranular cells, or the basal lamina, are overwhelmingly occupied by secretory granules as compared with the cytoplasm of secretory cells in the interior of the pars intermedia. The density of these granules varies considerably in the material prefixed with aldehydes, the smaller granules generally being denser. However, the granules are uniformly electron-lucent in the cytoplasmic processes of pars intermedia cells without prefixation (compare Fig. 10 and Fig. 11). Buddings of the granular membrane are found in a few secretory poles of pars intermedia cells. The diameters of these pars intermedia secretory granules are extremely variable from 2000 to 3000 A. Small "empty" vesicles with diameters 400 to 700 A are also abundantly scattered in the secretory pole of the cells. Dense bodies are occasionally found in this region. The ultrastructural organization of the boundary area between pars nervosa and pars intermedia is diagrammatically summarized in Fig. 12.

Ultrastructure of the Pars intermedia in Detail No structure that could be identified as a nerve fiber was found in the pars intermedia parenchyma. The cells of the pars intermedia are of two primary types: secretory cells and agranular, apparently non-secretory, cells. The cytoplasm of the non-secretory cells is meager in amount and variable in density (Fig. 10). It has poorly developed endoplasmic reticulum, small vesicles, irregular dense lamellated bodies, dense tiny particles and sometimes large colloid droplets. Chromatin appears accumulated near the nuclear membrane. Some of the non-secretory cells extend their cytoplasmic processes to the secretory surface of the pars intermedia as described above. The number of these cells is always much smaller in any part of the gland than that of secretory cells. Several states or differentiated regions of secretory cells arc discernible (Fig. 13): 1. Cells that have well developed, rough surfaced endoplasmic reticulum without prominent secretory granules (Fig. 14) ; 2. Cells that contain small dense 12"

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Fig. 12. Schematic representation of the boundary region between the pars nervosa and the pars intcrmedia of the adult lamprey. The width of perivascular space and capillary lumen is variable. The intrusion of nerve fibers into pars intermedia through this fibrovascular space is not encountered. An arrow indicates a possible synaptoid contact. B L basal lamina, CF collagen fibrils, C P capillary, D desmosome, D B dense body, E D endothelium, E P ependymal process, M mitochondria, NGC cytoplasmic process of non-granulated cell, P I pars intermedia, P N pars nervosa, P VS perivascular space, RC red blood cell, SG secretory granules in cell process of pars intermedia cell, I, I I , I I ' , I I I , I I I ' , and I V represent axons so designated

granules with d i a m e t e r s of 1600 to 2400 A with p r o m i n e n t limiting m e m b r a n e s as well as highly developed Golgi a p p a r a t u s a n d m o d e r a t e l y d e v e l o p e d endoplasmic r e t i c u l u m (Fig. 13). L u c e n t vesicles, l a m e l l a t e d bodies, dense r o u n d droplets a n d m i t o c h o n d r i a are also f r e q u e n t l y encountered. This cell t y p e is t h e c o m m o n e s t in t h e p a r s intermedia. 3. Cells with well developed, d i l a t e d endoplasmic reticuli, Golgi a p p a r a t u s , m i t o c h o n d r i a a n d small n u m b e r s of t h e granules similar to those in t h e t y p e - 2 cells (Fig. 13). 4. Cells similar to t h e t y p e - 3 cells,

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Fig. 13. Deeper region of the pars intermedia. Many secretory cells extend their cytoplasmic processes in a dorso-ventral direction. I I type 2 cell, I I I type 3 cell, I V type 4 cell, V I type 6 cell characterized by the intracisternal occurrence of large colloid droplets. Notice dilated endoplasmic reticular system in type 3 and 4 cells. Karnovsky's fixation, x 4400 Fig. 14. Type 1 cell in the pars intermedia. The cytoplasm is occupied by well developed endoplasmic reticulum. SG secretory granules in type 5 cells. OsOa fixation. • 8 700 Fig. 15. Colloid droplets in the profile of a type 5 cell. The content of the secretory granules seems to be discharged into the lumen (arrows) where dense colloid droplets float in the flocculent material. Karnovsky's fixation. • 11500

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but with particularly dense nucleus and cytoplasmic matrix (Fig. 13). 5. Cell profiles filled with large granules at their secretory poles as described earlier (Figs. 10, 11). 6. Cells with expanded endoplasmic reticuli in which large dense droplets with diameter of 1.5 to 2 ~ are present (Fig. 13). The number of these cells varies between individuals. Similar intracisternal droplets have been described in the pars intermedia cells of frogs, and their functional role discussed (for references, see Olsson and Gorbman, 1973). Cilia without prominent central tubules are often observed on the pars intermedia cells. These different cell "types" m a y in part represent secretory phases, or zonated stages of secretion of a single cell type. The following observations suggest this possibility. 1. Many secretory cells, if not all, extend their cytoplasmic secretory processes to the perivascular space on the dorsal surface of the pars intermedia, where they m a y correspond to the profiles of the ceils designated as the type 5. 2. The differentiation between type 1 and type 2 is not always possible, the former probably representing a very early stage of hormonal synthesis. 3. The t y p e 3 and 4 cells are not always found in every individual. The cytoplasmic organelles in these cells suggest a high level of synthetic activity or exhausted state resulting from it, which might follow the early synthetic stage represented by the type-2 cells. The similarity in the size of granules contained in them implies t h a t these cells belong to one type. 4. The large droplet characterizing the type-6 cells is occasionally found in the dilated endoplasmic reticulum in the type-5 cells (Fig. 15). Furthermore, the possibly early developmental stages of intracisternal droplet formation are followed in these cells. 5. Almost all cells stain with aldehyde fuchsin, although the degree of stainability varies among them. The poorly differentiated cells, actively synthetic cells and those laden with secretory granules, facing the fibrovascular septum between pars nervosa and pars intermedia, were also described for adult Lampetra /luviatilis (Larsen and Rothwell, 1972). Discussion

Hypothalamic Control of the Pars intermedia Although the contact of nerve fibers with secretory cells of the pars intermedia was described at the electron-microscopical level in ammocoete larvae of Lampetra planeri (Miiller, 1965), such a structural feature was not found by us in adult Lampetra tridentata. Green and Maxwell (1959) described, by use of light microscopy, a single possible nerve fiber entering the pars intermedia of the lamprey. However, other authors have failed to find any penetration by nerve fibers to pars intermedia in Lampetra planeri and Lampetra ]luviatilis (Bargmann, 1953; van de K a m e r and Schreurs, 1959; ])odd and Kerr, 1963; Rfihle and Sterba, 1966). Thus, the penetration of nerve axons into the pars intermedia of the adult lampreys is extremely rare, if it occurs at all. This implies that innervation plays no role, or a very minor one, in hypothalamic regulation of the secretory activity of the pars intermedia. On the other hand, the presence of an obviously active and secretory pars intermedia, its anatomical disposition as a relatively thin sheet of cells over a

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similarly shaped pars nervosa, argues that unless the spatial relationship is accidental, there m a y be an adaptive basis for it. The broad appositional anatomical relationship between nervous and glandular tissues, separated only by a pericapillary space, small capillaries and some collagenous fibers, might suggest a type of hypothalamic control over release of an MSH-like hormone in the adult lamprey that depends upon diffusion of presumptive chemical factor(s). I f the anatomical relationship between pars nervosa and pars intermedia thus has a functional value, and this value is related to nervous regulation over secretion of a hormone from the pars intermedia, then we must still explain how regulatory factor(s) reaches all of the cells of a pars intermedia that is avascular. Many secretory cells, if not all, seem to extend their elongated cytoplasmic processes to the septal surface, thus making contact with a neurogenic diffusing factor possible. The long cytoplasmic processes of the non-secretory cells, which might be designated "stellate cells", because they resemble cells so named in pars intermedia of higher vertebrates, might also transport regulatory factors to secretory cells in the interior of the pars intermedia. That some kind of neurogenic control exists m a y be supported by the fact t h a t removal of lateral eyes caused skin darkening in metamorphosed Mordacia and adult Lampetra, while the extirpation of the pineal had no effect on diurnal color change (Eddy and Strahan, 1968 ; Joss, 1973). These experiments seemingly suggest that the putative factor(s) involved between photic reception by the eye and a secretory response in the pars intermedia cells is inhibitory in the nature. Absorption of any presumptive released pars intermedia hormone apparently occurs from the dorsal and ventral surfaces of the pars intermedia, where the only blood capillaries are present. The high degree of stainability and the accumulation of m a n y mature secretory granules in the cells and cell processes near these capillaries fits this hypothesis. The participation of stellate cells in the release mechanism of pars intermedia hormone is also conceivable. The filamentous structures in the cytoplasmic processes of stellate cells could play a role in the transport of biologically active substances, as in the case of ependymal cells (see below). Dense bodies similar to lysosomes in the stellate cells also suggest a digestive function of stellate cells for hydrolytic activation of a possible MSH prohormone, or for destructive inactivation of MSH. Stellate cells or similar non-secretory intermedial cells, which are known by various names, occur quite frequently in various vertebrates (Nakai and Gorbman, 1969; Rodriguez and La Pointe, 1970; Weatherhead, 1971; Cameron and Foster, 1971; Forbes, 1972), and their functional properties or significance remain a matter for debate. In the absence of direct innervation of the pars intermedia, the adult lamprey is similar to the lizard, Klauberina riversiana (Rodriguez and La Pointe, 1970), Sphenodon punctatus (Weatherhead, 1971) and the sturgeon (Polenov et al., 1972). In the lizard, according to integumentary chromatic responses after hypophysial stalk section, it has been suggested that a central nervous influence is exerted on the pars intermedia cells and body color indirectly, and t h a t it is less effective and slower than in systems t h a t provide direct innervation of the pars intermedia, as in the frog (Rodriguez et al., 1971). The neuronal regulation of pars intermedia cells without direct innervation was also suggested in the lizard, Calotes versicolor (Pandalai and Sheela, 1969).

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Direct synaptoid innervagion of secretory cells of the pars intermedia is well described in elasmobranchs (Chevins, 1972), teleosts (Follenius, 1965; Vollrath, 1967; Kasuga and Takahashi, 1970; Leagherland, 1970; Zambrano, 1970), the newt (Dent and Gupta, 1967), anurans (Saland, 1968 ; Doerr-Schogt and Follenius, 1970; Igo, 1971; Nakai and Gorbman, 1969) and in mammals (Bargmann st al., 1967 ; Wittkowski, 1967 ; Vincent and Anand Kumar, 1969 ; Belenky st al., 1970; Baumgarten et al., 1972). Although synaptoid contacts were not found, the invasion of neurosecretory fibers into the pars ingermedia parenchyma was also described in the polypteriformid fish, Calamoichthys (Lagios, 1968), in some teleosts (Follenius, 1965; Knowles and Vollrath, 1966; Vollrath, 1967; Bern et al., 1971) as well as in the lungfish, Lepidosiren (Zambrano and Iturriza, 1973). It is difficult at this time to interpret the various degrees of intimacy of anatomical relationship between pars nervosa and pars ingermedia in various vertebrates, one extreme being represented by the lamprey, sturgeon and lizard, the other by the elasmobranehs and teleosts. There is no obvious phylogenetie or ecologic correlation between the presence or absence of such innervagion and the abundance of melanophores or the need for adaptive integumentary chromatic regulation. Recently, participation by ependymal cells of the neurohypophysis in the regulation of adenohypophysial function has been suggested for various vertebrates (Vigh st al., 1963; Knowles and Anand Kumar, 1969; Kobayashi et al., 1972). The fact that ependymal cells of the lamprey's pars nervosa manifest secretory and/or absorptive characteristics such as microvilli, pinoeytotic vesicles, large colloid droplets etc., implies a possible functional significance of ependymal cells in regulation of the adjacent pars ingermedia, relating the third ventricle to the blood capillaries embedded in the perivascular space. The possibility that ependymal cells relate function of intermedial cells with the pineal, which is known as the source of a skin-paling substance, through cerebrospinal fluid, and thus participates in the mechanism of body color change, is not excluded, bug it is purely speculative. Dense lamellaged bodies in ependymal cells might correlate with the hydrolysis of biologically active substances. A functional relationship between ependymal cells in the pars nervosa and the liquor-contacting ventricular processes of the neurosecretory cells of the preoptic nucleus through cerebrospinal fluid is also conceivable, as suggested in the ammocoete larva (Sterba and Briickner, 1967). The mechanism for regulating change in body color in the ammocoete seems to be different from that of the adult lamprey. The absence of the external eyes in the ammocoete suggests the utilization of a non-retinal photoreceptor. Experimental data indicating a function of the pineal in the change of body color has been provided for ammoeoeges of Geotria australia by Eddy and Strahan (1968) and for those of Lampetra by Young (1935) and Joss (1973). The fact that direct contact between nerve fibers and ingermedial cells has been described in larval lampreys (Mtiller, 1965) may also indicate that a different mechanism for chromagophore control exists in the ammocoete. Ultrastructure of the Pars nervosa The functional significance of the various kinds of granules and vesicles found in the axonal terminals of the lamprey pars nervosa would be difficult go delineate

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a t this time. Of t h e k n o w n possibilities, these granules a n d vesicles could r e p r e s e n t n e u r o h y p o p h y s i a l o c t a p e p t i d e s a n d m o n o a m i n e s t o g e t h e r with t h e i r carriers, a n d also t h e y could r e p r e s e n t different phases of secretion or d e p l e t i o n of such materials. Arginine vasotocin is t h e o n l y n e u r o h y p o p h y s i a l o c t a p e p t i d e identified from t h e l a m p r e y ( S a w y e r et al., 1961; F o l l e t t a n d Heller, 1964). T h e association of arginine vasotocin with t h e granules has been suggested in t h e teleost fish (Lederis, 1962). The presence of a t least two t y p e s of electron-dense granules, one with a d i a m e t e r a b o u t 1800 A a n d t h e other a b o u t 1200 A, suggests t h a t t h e r e are a t least two k i n d s of peptidergic n e u r o s e c r e t o r y m a t e r i a l s in t h e l a m p r e y . The axons containing these granules m i g h t correspond, respectively, to t h e t y p e A1 a n d A2 axons in t h e s h a r k (Knowles, 1965) a n d teleosts (Knowles a n d Vollrath, 1966). T h e electron-dense granules, a b o u t 700 to 800/~ in d i a m e t e r , are generally r e g a r d e d as m o n o a m i n e granules using criteria p r o v i d e d b y t h e a p p l i c a t i o n of various m e t h o d s (see Z a m b r a n o et al., 1972). T h e p a u c i t y of t y p e - I I fibers in t h e present m a t e r i a l is consistent with t h e weak m o n o a m i n e oxidase a c t i v i t y f o u n d in t h e p a r s nervosa of Lampetra japonica (Tsuneki, 1974). Since we h a v e been considering a possible n e u r o s e c r e t o r y contol of t h e p a r s i n t e r m e d i a b y diffusion of a h y p o t h e t i c a l M S H - R F or M S H - R I F , t h e n c o n c e i v a b l y one or m o r e t y p e s of t h e visible granules we have described in t h e p a r s nervosa of t h e l a m p r e y m a y be r e l a t e d to this function.

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Ultrastructure of pars nervosa and pars intermedia of the Lamprey, Lampetra tridentata.

The pars intermedia of the adult lamprey is separated by perivascular spaces and a capillary plexus from the pars nervosa. No penetration of nerve fib...
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