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Fine structure of the neurohemal areas associated with the hypophysis in the hagfish, Polistotrema stoutii
GENERAL
AND
COMPAR.4TIVE
ENDOCRINtiOGY
7,
457-462
(1966)
Fine Structure of the Neurohemal Hypophysis in the Hagfkh, RICHARD Department
S. NISHIOKA of Zoology University
Areas Associated with Polistotrema sfoutii AND
HOWARD
and Its Cancer Research of Calijornia, Bexkeley,
Received
March
the
A. BERN
Genetics California
Laboratory,
14, 1966
The structure of an anterior neurohemal area in the chiasmatic region of the diencephalic floor in the hagfish, Polistotrema stoutii, has been studied by light and electron microscopy, and compared with that of the neurohypophysis. This region, from which blood is carried to the neurohypophysis by a port.al system described first by Gorbman and collaborators, has much in common with the median eminence of tetrapods: location, paraldehyde-fuchsin stainability, and axon terminals containing small vesicles, despite the absence of a relation with the adenohypophysis.
Gorbman et al. (1963) described a portal system of unknown significance leading from the anterior diencephalic floor to the neurohypophysis in the hagfish, Polistotrema stoutii. The point of origin of this system, called the chiasmatic region herein, stains with paraldehyde fuchsin and is strongly reminiscent of the tetrapod median eminence. The present study was undertaken to examine the nature of this anterior hypothalamic neurohemal area and to compare it with the structure of the hagfish neurohypophysis. MATERIALS
AND
METHODS
Hagfish, Polistotrema stoutii, were caught in Monterey Bay, California. Twelve animals between 20 and 24 inches in length were used in the study. The brains of seven animals were exposed and fixed in Bouin’s fluid. Paraffin sections were stained with paraldehyde fuchsin and counterstains. The chiasmatic region and the neurohypophysis of five animals were removed and fixed in phosphate-buffered glutaraldehyde followed by impregnation with osmium tetroxide. The tissues were embedded in Maraglas-Cardolite, and sections were picked up on Formvar-coated grids backed “stained” with uranium acetate folwith carbon, lowed by lead citrate, and examined in an RCA EMU 3F electron microscope.
OBSERVATIONS
The chiasmatic region is characterized by hazy paraldehyde-fuchsin staining, some prominent Herring bodies, and a dist’inct capillary plexus (Fig. 1). It is a bilaterally symmetrical area; the major staining areas are located laterally but a light fuchsinophilic zone crossesthe midline. Occasionally more than one locus of fuchsinophilic staining and capillary plexus can be found laterally. The “prehJipophysia1 plexus” is formed by dorsally projecting capillaries from the prehypophysial artery, a branch of the ventral internal carotid artery which courses toward the midline and which lies on the floor of the brain (Gorbman et al., 1963). Thus, the major part of the chiasmatic region was usually anterodorsal and adjacent to the artery (Fig. 1). Dorsal and medial to this region, both rostrallp and caudally, a few neurons with fuchsinophiiic, material were observed. Some nerve fibers were also faintly fuchsinophilic, but t)hey could not be traced for any appreciable distance. Fewer capillaries per unit area were seen in the central region than in the lateral and more superficial chiasmatic region. 457
458
KISHIOGA
AND
BERN
FIG. 1. Sagittal section of chiasmatic region with darkly stained fuchsinophilic material around capillaries of prehypophysial plexus. PHA, prehypophysial artery. x 250. FIG. 2. Midsagittal section of neurohypophysis. Fuchsinophilic material is adjacent to capillaries located on dorsal wall of infundibular process. Thick connective tissue lying below layer of ependyma forms ventral wall of infundibular process. Adenohypophysis (not shown) is located ventrally and is separated from neurohypophysis by thick connective tissue. x 100. FIG. 3. Higher magnification of fuchsinophilic area of dorsal neurohypophysial wall shown in Fig. 2. Note unstained ependyma and darkly stained fuchsinophilic material adjacent to capillary lumen. X 250.
The histology of the hagfish neurohypophysis has been described by several workers (e.g., Adam, 1959, 1963; Olsson, 1959; Matty, 1960). In brief, the infundibular process is a dorsoventrally compressed, saccular structure with the stalk located near its anterior end (Fig. 2). Most of the
fuchsinophilic material is located in the posterior part of the dorsal wall between the ependyma and capillaries, closely applied to the latter (Fig. 3). A network of capillaries covers the outer surface of the dorsal wall. Very little if any fuchsinophilic material is present in the ventral wall and
SEUROHEMAL
AREAS
OF
HAGFISH
BRAIX
459
in the anterior part of the dorsal wall, which cipitates were also evident in some axons, are both composed of a thin layer of presumably on glycogen deposits. The basement, membrane region of the ependyma covered by connective tissue. No dense elementary neurosecretory capillary was not thick, averaging 250011; however, unlike that of the chiasmatic granules were found in the axons bordering capillaries, collagen fibrils were embedde on capillaries in the paraldehyde fuchsinstairring chiasmatic region (Figs. 4 and 5). in the ground substance (Fig. 6). Many pinocyt.ic vesicles were present in the endoThe few large axons adjacent to capillaries contained only small vesicles, 300-500 B in thelium, some opening at the lumen side and diameter. Some vesicle-containing axons others at the basement membrane side. were more dense than others. Part of their DISCUSSfON high density may be attributed to the close The enigmatic nature of the portal system packing of small vesicles of uniform size. Denser axoplasmic ground substance may described by Gorbman et at. (1963) as is also be a contributing factor. The less dense carrying blood to the neurohypophysis further emphasized by the detailed ultraaxons contained fewer vesicles, of variable The primary capilsizes and often of irregular shapes. The structural information. axons of the chiasmatic region contained lary plexus unquestionably takes its origin more mitochondria than did the axons of in a true neurohemal area in the diencethe neurohypophysis. Precipitation of lead phalic floor in the region of the optic salts, in part on glycogen deposits, was chiasma. We are uncertain as to the existprominent in some glial processes and axons. ence of a neurosecretory “nucleus postMany synapse-like junctions between opticus” as described by Gorbman et ai. nerve fibers were observed (Figs. 4 and 5). (1963), but there is no doubting the nature More of these junctions appeared to occur of the axon-capillary relations, which define between pale and dense axons than’between this system as neurosecretory. axons of the same type. The axons did not Of particular interest is the emergence in end directly on the thin capillary basement this cyclostome fish of an ultrastructural membrane (about 800 A thick) but were distinct.ion between anterior (chiasmatic) separated from it by a layer of dense glial and posterior (neurohypophysial) neurocells. hemal areas virtually parallel to that seen In the neurohypophysis, axons containing in tetrapods. The median eminence and the membrane-limited, electron-dense granules pars nervosa of at least some tetrapods reand small vesicles, as well as axons filled cently studied are clearly distinct in the only with vesicles, abut on the capillaries nature of the inclusions in the axon termi(Fig. 6). IJsually granules (100&2OOOA) nals. Median eminence terminals in the were found in the larger axons, and vesicles amphibian 1’T.yla (Smaller, 1966) and in the in the smaller axons, but many fibers of all bird Zonotrichia (Bern et aE., 1966; see Bern sizes contained granules and vesicles. The and Nishioka, 1965, for observations ‘on granules were variable in density ; the palest other passerine birds) are chara,cterized by ones were really large vesicles. Despite the the presence of large numbers of srn$ wide range of vesicle sizes, the great majorvesicles reminiscent of synaptic vesicles. ity were small and measured between 300 Pars nervosa terminals, on the other hand: and 5OOA in diameter. show rmmerous typical elementary neuroMost of the axons adjacent to the capilsecretory granules. Both neurohemal areas laries were separated from the basement in tet.rapods stain with paraldehyde iuchsin. membrane by a glial or ependymal layer. In the hagfish similarly, the anterior neuroMost of the axons were also separated from hernal area is charac,terized by the presence each other by glial or ependymal processes. of numerous small vesicles and the,poste&or Many of these processes contained fine neurohypophysis by many electron-dense fibrils, upon which a considerable amount of granules. Again, both areas stain ‘With parlead salt was precipitated. Lead salt pre- aldehyde fuchsin.
FIG. 4. Electron micrograph of a p&capillary region in prehypophysial plexus in chiasmatic region. Axons containing small vesicles abut on glial cell processes. Several pale axons (PA) contain fewer and often more irregular vesicles than dark axons (DA). Arrows indicate possible synaptic structures. Basement membrane (BM) is dense and thin, apparently containin g no collagen fibrils. RBC, erythrocyte; EN, endothelium.
FIG, 5. Similar region as in Fig. 4. Note one pale axon (PA) with two dark axons (DA) and another pale axon. BM, basement ‘erythrocyte. 461
in possible membrane;
synaptic relation EN, endothelium;
(arrows) RBC,
462
NISHIOKA
In many respects it is tempting to refer to the chiasmatic region in the hagfish as a “median eminence”: it is in a comparable anatomic location ; it is a neurohemal area ; it is the source of a portal system; it is paraldehyde fuchsin-staining; and it is replete with small vesicles in its axon terminals, whose origin is uncertain. Olsson (1959) and Adam (1963) have described as a “median eminence” in Myxine a neurohemal area just anterior to the neurohypophysial stalk, which does not seem to be in the same location as in Polistotpema. The question of the significance of a system portal to the neurohypophysis instead of the adenohypophysis has been raised by its discoverers (Gorbman et al., 1963; Gorbman, 1965). It is possible that the neurohormones secreted into the primary portal plexus serve as blood-transported triggers or inhibitors to the release of secretion by the neurohypophysis. However, fibers with small vesicles are also present, in the neurohypophysis, and these could serve as sources of regulators of secretion discharge by the neurosecretory axons proper (cf. Bern, 1962). In any case, the new information presented herein casts further doubt on the validity of Wingstrand’s (1959) suggestion that the primitive neurohypophysis has a median eminence-like (adenohypophysisregulating) function. The definitive neurohypophysis of the hagfish resembles closely the pars nervosa of tetrapods ultrastructurally; the considerably less prominent chiasmatic neurohemal area resembles the median eminence of tetrapods ultrastructurally. ACKNOWLEDGMENTS We are grateful to David Evans, Schroeder, and the Hopkins Marine Station aid in t.he acquisition of hagfish material.
Paul for This
AND
work NSF
BERN
was supported by research grants from (GB-2484) and the NIH (CA-070815).
the
REFERENCES ADAM, H. (1959). Hypophyse und hypothalamoneurohypophyskes Neurosekretsystem bei den Cyclostomen Mgxine glutinosa und Bdellostoma stouti. Verhandl. Deutsch. Zool. Gesell. 18, 157171. ADAM, H. (1963). The pituitary gland. In “The Biology of Myxine” (A. Brodal and R. Fange, eds.), pp. 459476. Universitetsforlaget, Oslo. BERN, H. A. (1962). Properties of neurosecretory cells. Gen. Comp. Endocrinol. 1, 117-132. BERN, H. A., AND NISHIOKA, R. S. (1965’). Fine structure of the median eminence of some passerine birds. Proc. Zool. Sot. Calcutta 18, 107119. BERN, H. A., NISHIOKA, R. S., MEWALDT, L. R., AND FARNER, D. S. (1966). Photoperiodic and osmotic influences on the ultrastructure of the hypothalamic neurosecretory system of the White-crowned Sparrow, Zonotrichia leucophrys gambelii. Z. Zellforsch. 69, 19&227. GORBMAN, A. (1965). Vascular relations between the neurohypophysis and adenohypophysis of cyclostomes and the problem of evolution of hypothalamic neuroendocrine control. Arch. d’rlnat. Micros. 54, 163-194. GORBMAN, A., KOBAYASHI, H., AND UEMURA, H. (1963). The vascularization of the hypophysial structures of the hagfish. Gen. Camp. Endocrinol. 3, 505514. MATTY, A. J. (1960). The pituitary of Myxine glutinosa. Nature 18.5, 180-181. OLSS~N, R. (1959). The neurosecretory hypothalamus system and the adenohypophysis of Myxine. Z. Zellforsch. 51, 97-107. SMOLLER, C. G. (1966). Ultrastructural studies on the developing neurohypophysis of the Pacific treefrog, Hyla regilla. Gen. Comp. Endocrinol. 7, 44-73. WINGSTRAND, K. G. (1959). Attempts at a comparison between the neurohypophysial region in fishes and tetrapods, with particular regard to amphibians. Zn “Comparative Endocrinology” (A. Gorbman, ed.), pp. 393-401. Academic Press, New York.
AND
COMPAR.4TIVE
ENDOCRINtiOGY
7,
457-462
(1966)
Fine Structure of the Neurohemal Hypophysis in the Hagfkh, RICHARD Department
S. NISHIOKA of Zoology University
Areas Associated with Polistotrema sfoutii AND
HOWARD
and Its Cancer Research of Calijornia, Bexkeley,
Received
March
the
A. BERN
Genetics California
Laboratory,
14, 1966
The structure of an anterior neurohemal area in the chiasmatic region of the diencephalic floor in the hagfish, Polistotrema stoutii, has been studied by light and electron microscopy, and compared with that of the neurohypophysis. This region, from which blood is carried to the neurohypophysis by a port.al system described first by Gorbman and collaborators, has much in common with the median eminence of tetrapods: location, paraldehyde-fuchsin stainability, and axon terminals containing small vesicles, despite the absence of a relation with the adenohypophysis.
Gorbman et al. (1963) described a portal system of unknown significance leading from the anterior diencephalic floor to the neurohypophysis in the hagfish, Polistotrema stoutii. The point of origin of this system, called the chiasmatic region herein, stains with paraldehyde fuchsin and is strongly reminiscent of the tetrapod median eminence. The present study was undertaken to examine the nature of this anterior hypothalamic neurohemal area and to compare it with the structure of the hagfish neurohypophysis. MATERIALS
AND
METHODS
Hagfish, Polistotrema stoutii, were caught in Monterey Bay, California. Twelve animals between 20 and 24 inches in length were used in the study. The brains of seven animals were exposed and fixed in Bouin’s fluid. Paraffin sections were stained with paraldehyde fuchsin and counterstains. The chiasmatic region and the neurohypophysis of five animals were removed and fixed in phosphate-buffered glutaraldehyde followed by impregnation with osmium tetroxide. The tissues were embedded in Maraglas-Cardolite, and sections were picked up on Formvar-coated grids backed “stained” with uranium acetate folwith carbon, lowed by lead citrate, and examined in an RCA EMU 3F electron microscope.
OBSERVATIONS
The chiasmatic region is characterized by hazy paraldehyde-fuchsin staining, some prominent Herring bodies, and a dist’inct capillary plexus (Fig. 1). It is a bilaterally symmetrical area; the major staining areas are located laterally but a light fuchsinophilic zone crossesthe midline. Occasionally more than one locus of fuchsinophilic staining and capillary plexus can be found laterally. The “prehJipophysia1 plexus” is formed by dorsally projecting capillaries from the prehypophysial artery, a branch of the ventral internal carotid artery which courses toward the midline and which lies on the floor of the brain (Gorbman et al., 1963). Thus, the major part of the chiasmatic region was usually anterodorsal and adjacent to the artery (Fig. 1). Dorsal and medial to this region, both rostrallp and caudally, a few neurons with fuchsinophiiic, material were observed. Some nerve fibers were also faintly fuchsinophilic, but t)hey could not be traced for any appreciable distance. Fewer capillaries per unit area were seen in the central region than in the lateral and more superficial chiasmatic region. 457
458
KISHIOGA
AND
BERN
FIG. 1. Sagittal section of chiasmatic region with darkly stained fuchsinophilic material around capillaries of prehypophysial plexus. PHA, prehypophysial artery. x 250. FIG. 2. Midsagittal section of neurohypophysis. Fuchsinophilic material is adjacent to capillaries located on dorsal wall of infundibular process. Thick connective tissue lying below layer of ependyma forms ventral wall of infundibular process. Adenohypophysis (not shown) is located ventrally and is separated from neurohypophysis by thick connective tissue. x 100. FIG. 3. Higher magnification of fuchsinophilic area of dorsal neurohypophysial wall shown in Fig. 2. Note unstained ependyma and darkly stained fuchsinophilic material adjacent to capillary lumen. X 250.
The histology of the hagfish neurohypophysis has been described by several workers (e.g., Adam, 1959, 1963; Olsson, 1959; Matty, 1960). In brief, the infundibular process is a dorsoventrally compressed, saccular structure with the stalk located near its anterior end (Fig. 2). Most of the
fuchsinophilic material is located in the posterior part of the dorsal wall between the ependyma and capillaries, closely applied to the latter (Fig. 3). A network of capillaries covers the outer surface of the dorsal wall. Very little if any fuchsinophilic material is present in the ventral wall and
SEUROHEMAL
AREAS
OF
HAGFISH
BRAIX
459
in the anterior part of the dorsal wall, which cipitates were also evident in some axons, are both composed of a thin layer of presumably on glycogen deposits. The basement, membrane region of the ependyma covered by connective tissue. No dense elementary neurosecretory capillary was not thick, averaging 250011; however, unlike that of the chiasmatic granules were found in the axons bordering capillaries, collagen fibrils were embedde on capillaries in the paraldehyde fuchsinstairring chiasmatic region (Figs. 4 and 5). in the ground substance (Fig. 6). Many pinocyt.ic vesicles were present in the endoThe few large axons adjacent to capillaries contained only small vesicles, 300-500 B in thelium, some opening at the lumen side and diameter. Some vesicle-containing axons others at the basement membrane side. were more dense than others. Part of their DISCUSSfON high density may be attributed to the close The enigmatic nature of the portal system packing of small vesicles of uniform size. Denser axoplasmic ground substance may described by Gorbman et at. (1963) as is also be a contributing factor. The less dense carrying blood to the neurohypophysis further emphasized by the detailed ultraaxons contained fewer vesicles, of variable The primary capilsizes and often of irregular shapes. The structural information. axons of the chiasmatic region contained lary plexus unquestionably takes its origin more mitochondria than did the axons of in a true neurohemal area in the diencethe neurohypophysis. Precipitation of lead phalic floor in the region of the optic salts, in part on glycogen deposits, was chiasma. We are uncertain as to the existprominent in some glial processes and axons. ence of a neurosecretory “nucleus postMany synapse-like junctions between opticus” as described by Gorbman et ai. nerve fibers were observed (Figs. 4 and 5). (1963), but there is no doubting the nature More of these junctions appeared to occur of the axon-capillary relations, which define between pale and dense axons than’between this system as neurosecretory. axons of the same type. The axons did not Of particular interest is the emergence in end directly on the thin capillary basement this cyclostome fish of an ultrastructural membrane (about 800 A thick) but were distinct.ion between anterior (chiasmatic) separated from it by a layer of dense glial and posterior (neurohypophysial) neurocells. hemal areas virtually parallel to that seen In the neurohypophysis, axons containing in tetrapods. The median eminence and the membrane-limited, electron-dense granules pars nervosa of at least some tetrapods reand small vesicles, as well as axons filled cently studied are clearly distinct in the only with vesicles, abut on the capillaries nature of the inclusions in the axon termi(Fig. 6). IJsually granules (100&2OOOA) nals. Median eminence terminals in the were found in the larger axons, and vesicles amphibian 1’T.yla (Smaller, 1966) and in the in the smaller axons, but many fibers of all bird Zonotrichia (Bern et aE., 1966; see Bern sizes contained granules and vesicles. The and Nishioka, 1965, for observations ‘on granules were variable in density ; the palest other passerine birds) are chara,cterized by ones were really large vesicles. Despite the the presence of large numbers of srn$ wide range of vesicle sizes, the great majorvesicles reminiscent of synaptic vesicles. ity were small and measured between 300 Pars nervosa terminals, on the other hand: and 5OOA in diameter. show rmmerous typical elementary neuroMost of the axons adjacent to the capilsecretory granules. Both neurohemal areas laries were separated from the basement in tet.rapods stain with paraldehyde iuchsin. membrane by a glial or ependymal layer. In the hagfish similarly, the anterior neuroMost of the axons were also separated from hernal area is charac,terized by the presence each other by glial or ependymal processes. of numerous small vesicles and the,poste&or Many of these processes contained fine neurohypophysis by many electron-dense fibrils, upon which a considerable amount of granules. Again, both areas stain ‘With parlead salt was precipitated. Lead salt pre- aldehyde fuchsin.
FIG. 4. Electron micrograph of a p&capillary region in prehypophysial plexus in chiasmatic region. Axons containing small vesicles abut on glial cell processes. Several pale axons (PA) contain fewer and often more irregular vesicles than dark axons (DA). Arrows indicate possible synaptic structures. Basement membrane (BM) is dense and thin, apparently containin g no collagen fibrils. RBC, erythrocyte; EN, endothelium.
FIG, 5. Similar region as in Fig. 4. Note one pale axon (PA) with two dark axons (DA) and another pale axon. BM, basement ‘erythrocyte. 461
in possible membrane;
synaptic relation EN, endothelium;
(arrows) RBC,
462
NISHIOKA
In many respects it is tempting to refer to the chiasmatic region in the hagfish as a “median eminence”: it is in a comparable anatomic location ; it is a neurohemal area ; it is the source of a portal system; it is paraldehyde fuchsin-staining; and it is replete with small vesicles in its axon terminals, whose origin is uncertain. Olsson (1959) and Adam (1963) have described as a “median eminence” in Myxine a neurohemal area just anterior to the neurohypophysial stalk, which does not seem to be in the same location as in Polistotpema. The question of the significance of a system portal to the neurohypophysis instead of the adenohypophysis has been raised by its discoverers (Gorbman et al., 1963; Gorbman, 1965). It is possible that the neurohormones secreted into the primary portal plexus serve as blood-transported triggers or inhibitors to the release of secretion by the neurohypophysis. However, fibers with small vesicles are also present, in the neurohypophysis, and these could serve as sources of regulators of secretion discharge by the neurosecretory axons proper (cf. Bern, 1962). In any case, the new information presented herein casts further doubt on the validity of Wingstrand’s (1959) suggestion that the primitive neurohypophysis has a median eminence-like (adenohypophysisregulating) function. The definitive neurohypophysis of the hagfish resembles closely the pars nervosa of tetrapods ultrastructurally; the considerably less prominent chiasmatic neurohemal area resembles the median eminence of tetrapods ultrastructurally. ACKNOWLEDGMENTS We are grateful to David Evans, Schroeder, and the Hopkins Marine Station aid in t.he acquisition of hagfish material.
Paul for This
AND
work NSF
BERN
was supported by research grants from (GB-2484) and the NIH (CA-070815).
the
REFERENCES ADAM, H. (1959). Hypophyse und hypothalamoneurohypophyskes Neurosekretsystem bei den Cyclostomen Mgxine glutinosa und Bdellostoma stouti. Verhandl. Deutsch. Zool. Gesell. 18, 157171. ADAM, H. (1963). The pituitary gland. In “The Biology of Myxine” (A. Brodal and R. Fange, eds.), pp. 459476. Universitetsforlaget, Oslo. BERN, H. A. (1962). Properties of neurosecretory cells. Gen. Comp. Endocrinol. 1, 117-132. BERN, H. A., AND NISHIOKA, R. S. (1965’). Fine structure of the median eminence of some passerine birds. Proc. Zool. Sot. Calcutta 18, 107119. BERN, H. A., NISHIOKA, R. S., MEWALDT, L. R., AND FARNER, D. S. (1966). Photoperiodic and osmotic influences on the ultrastructure of the hypothalamic neurosecretory system of the White-crowned Sparrow, Zonotrichia leucophrys gambelii. Z. Zellforsch. 69, 19&227. GORBMAN, A. (1965). Vascular relations between the neurohypophysis and adenohypophysis of cyclostomes and the problem of evolution of hypothalamic neuroendocrine control. Arch. d’rlnat. Micros. 54, 163-194. GORBMAN, A., KOBAYASHI, H., AND UEMURA, H. (1963). The vascularization of the hypophysial structures of the hagfish. Gen. Camp. Endocrinol. 3, 505514. MATTY, A. J. (1960). The pituitary of Myxine glutinosa. Nature 18.5, 180-181. OLSS~N, R. (1959). The neurosecretory hypothalamus system and the adenohypophysis of Myxine. Z. Zellforsch. 51, 97-107. SMOLLER, C. G. (1966). Ultrastructural studies on the developing neurohypophysis of the Pacific treefrog, Hyla regilla. Gen. Comp. Endocrinol. 7, 44-73. WINGSTRAND, K. G. (1959). Attempts at a comparison between the neurohypophysial region in fishes and tetrapods, with particular regard to amphibians. Zn “Comparative Endocrinology” (A. Gorbman, ed.), pp. 393-401. Academic Press, New York.