Cell Tiss. Res. 166, 511-520 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
Neurosecretory Cell Types in the Eyestalk of the Freshwater Prawn Palaemon paucidens An Electron Microscopic Study* Setsuji Hisano Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan
Summary. In the medulla terminalis ganglionic X-organ (MTGX) of the eyestalk of the freshwater prawn, Palaemon paucidens, six peptidergic neurosecretory cell types (A-, B-, C-, D-, E-, and F-cells) are distinguishable on the basis of the different morphology of their elementary granules and rough endoplasmic reticulum (rER). All of these cell types seem to correspond to Type-IIIa cells or dispersing Type-IV cells, that have previously been differentiated at the light microscopic level (Hisano, 1974), as judged from the dimensions of their cell bodies and nuclei. Two other peptidergic neurosecretory cell types that are apparently comparable to the Type-II and Type-IIIb cells (Hisano, 1974), respectively, are recognized in parts of the optic ganglia other than MTGX, and these are now designated as G- and H-cells, respectively. All the remaining cell types, designated as Type-I, cluster-forming Type-IV, Type-V and Type-VI cells in our previous light microscopic study, have small cored-vesicles in their cytoplasm. It remains undecided whether these, possibly aminergic, neurons are neurosecretory or not.
Key words: Neurosecretory cells - Eyestalk - Palaemon - Ultrastrncture. Introduction In a previous study (Hisano, 1974), based on Gomori staining (chrome-hematoxylin phloxine, CHP; aldehyde fuchsin, AF) six types of possibly neurosecretory cells were identified in the optic ganglia of the freshwater prawn, Palaemon paucidens. However, these methods are selective rather than specific for neurosecretory products (Bern, 1962; Bern and Knowles, 1966; Gabe, 1966). Electron microscopy has been used in the present study to arrive at a more reliable Send offprint requests to: Dr. S. Hisano, Zoological Institute, Faculty of Science, Hokkaido Univer-
sity, Sapporo, 060 Japan. * The author wishes to express his sincere appreciation to Prof. T. Aoto for his invaluable advice during the course of this study.
512
S. Hisano
identification. In vertebrates and insects neurosecretory neurons are, as a rule, characterized by electron-dense granules, approximately 500-3,000 A in diameter (Bargmann, 1966; Knowles, 1967; Scharrer, 1969). The same holds for crustaceans.
Material and Methods For electron microscopy only large female Palaemon paucidens, at the intermolt period, were used. Optic ganglia were placed in ice-cold 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) containing 7% sucrose, and fixed in renewed fixative for about 2 hrs at (L4 ~ C. The specimens were postfixed in 1% OsO4 solution in the same buffer for 1-2 hrs at 0 4 ~ C and embedded in Epon 812 (Luft, 1961). Ultra-thin sections were stained with lead citrate (Reynolds, 1963), and examined in a Hitachi HS-7 electron microscope.
Results In the medulla terminalis ganglionic X-organ (MTGX), six types of neurosecretory neurons were identified, based on different features of their elementary granules and rER. These neurons seem to correspond to the cells previously identified light microscopically as Type-IIIa or as dispersing Type-IV cells (Hisano, 1974), as judged from the dimensions of the cell bodies and nuclei. These six cell types will now be referred to as A-, B-, C-, D-, E-, and F-cells, respectively (Table 1).
Table 1. Interrelation between neurosecretory cell types classified in the present and the previous studies Present study
Size of elementary granules or vesicles
Hisano (1974)
Remarks
Type-Ilia cell or Dispersing Type-IV cell
Peptidergic
(A) Elementary granules A-cell B-cell C-cell D-cell E-ceU F-cell
1,000~,300 1,000-2,100 1,000-1,700 700 1,400 700-1,300 700-1,300
G-cell
700-2,800
Type-II cell
Peptidergic
H-cell
800-1,200
Type-IIIb cell
Peptidergic
Type-I cell Cluster-forming Type-IV cell Type-V cell Type-VI cell
Aminergic (.9)
Vesicles 900-1,400 600-1,000 700-1,200 500- 900 a
(1,600)" (1,500) (1,300) (1,000) (1,000) (1,000)
(1,000) a (900) (900) (700)
Mean diameter shown in parentheses.
Crustacean Eyestalk Neurosecretory Cells
513
30-
J,J
,,=, 20,,=
',,,
i i
', ', ,.,..
==,
r
10-
0b0 1 a'oo GRANULEDIAMETER(,~)
2 o'oo
Fig. 1. Frequency distribution of diameters of elementary granules in six neurosecretory cell types of the M T G X . o - - o F-cell
A-cell; n - - i
B-cell; o - - - o
C-cell; o - - - o
D-cell; 9 . . . . . 9 E - c e l l ; o - - o
A - Cell
The not very abundant elementary granules of the A-cell are spheroidal and rather large (1,000-2,300 ,~; 1,600 A average, in diameter; Figs. 1, 2). A highly electron-dense matrix, homogeneous in appearance, is bounded by a thin limiting membrane. Most granules are dispersed throughout the cytoplasm. In the well developed Golgi complex the cisternae are occasionally enlarged at the ends, and contain a moderately dense material, apparently giving rise to the elementary granules. The rER consists of flattened cisternae running parallel to each other. The intra-cisternal space is slightly electron-dense owing to the presence of some flocculent material.
B-Cell
A relatively large number of elementary granules are present. The Golgi complex frequently contains electron-dense material in its cisternae (Fig. 3). The elementary granules, 1,000-2,100 A (average 1,500 A; Fig. 1), are round. Their electron density is lower than that of those in the A-cells. The rER consists of a number of short cisternae and vesicles with an electron-lucent and relatively wide lumen. Lysosome-like dense bodies of various sizes and shapes often contain a few
514
S. Hisano
Crustacean Eyestalk Neurosecretory Cells
515
elementary granules (Fig. 3, arrow). In a few cells with a number of semicircular subunits, there are large inclusions, 4-5 la in diameter (Fig. 4). The exact nature and functional significance, if any, of these inclusions are still unknown.
C-Ce// This cell has round or spheroidal medium-sized electron-dense elementary granules, approximately 1,000-1,700,~ (average 1,300A; Figs. 1, 5). Their number fluctuates considerably in relation to the morphology of the rER. In cells with cisternal or vesicular rER a large number of elementary granules are present, whereas in cells whose rER consists of distended sacs, only a few elementary granules are observed near the Golgi elements. These differences suggest different phases in the secretory cycle of the C-cells.
D-Ce// In the D-cell, the small elementary granules, approximately 700-1,400 A (average 1,000 A), are round (Figs. 1, 6). The cisternae of the rER have an electron-lucent, wide lumen. Golgi complexes are well developed, with often dense contents (Fig. 6, arrow).
E-Ce// In this cell, a small number of round elementary granules, 700-1,300 A (average 1,000 A) are present (Figs. 1, 7). The relatively abundant rER consists of slender parallel cisternae. The cytoplasm between cisternae is slightly more electrondense than the remaining cytoplasm (Fig. 7), due to the presence of flocculent material. Also found here and there in the cytoplasm are vacuoles of varying size and shape, and glycogen particles. The vacuoles always contain an anastomosing filamentous material whose functional role is unknown.
F-Ce// The elementary granules of this cell are round and small, measuring approximately 700-1,300 A (average 1,000 A; Fig. 1). They are electron-dense and scattered among the other organelles (Fig. 8). The content of the slender rER cister-
Fig. 2. Electron micrograph of A-cell containing highly electron-dense elementary granules. Note rER consisting of parallel cisternae and lysosome-like bodies (Ly). • 18,000 Fig. 3. B-cell with many elementary granules of moderate electron density. Note lysosome-like dense body (arrow) containing several elementary granules, x 20,700 Fig.4. Large inclusion body filled with numerous semicircular subunits in B-cell. x 9,100
516
S. Hisano
Fig. 5. C-cell with cisternal and vesicular rER. Elementary granules actively elaborated in Golgi complex (G). x 18,400 Fig. 6. Electron micrograph of D-cell. Note electron-dense content (arrow) in Golgi complex (G). • 16,100
Crustacean Eyestalk Neurosecretory Cells
517
nae is moderately electron-dense. Their distribution in the cytoplasm is disorderly.
Neurosecretory Cells of the Optic Ganglia other than the MTGX At least two additional types of neurons, located in the optic ganglia other than the MTGX, were found to be neurosecretory. These are now termed G-cell and H-cell; they were previously called Type-II and Type-IIIb (Hisano, 1974; see Table 1). G-Ce// The G-cell contains clumps of numerous elementary granules throughout the cytoplasm (Fig. 9), which seem to correspond to the masses of "Gomori-positive" material in light micrographs. The granules are round or spheroidal and range from 700 to 2,800 A. Most granules appear "homogeneous". Some show a coarse matrix of low electron density (Fig. 10, arrows). The usual limiting membrane of these granules is occasionally torn. Electron-dense material is always present in Golgi cisternae. The lamellar rER, for the most part, has an electron-lucent content. In some cells with coarse elementary granules of low electron density the intracisternal material is a little more dense. H-Ce// Elementary granules, approximately 800-1,200 A, are very scarce, although this cell contains abundant "Gomori-positive" material (Hisano, 1974). Most of the electron-dense granules lie near the Golgi complexes (Fig. 11). A large number of empty vesicles occur in the perinuclear region and at the cell periphery. These vesicles are round or oblong, varying from 700-1,900 A (average 1,300 .~). There still remains much conjecture as to the origin of these vesicles. Both rER and sER occur in this cell. The rER has the form of well-developed lamellae, and the sER is composed of closely-arranged tubules of fairly uniform width (Fig. 12). The role of the latter is unclear.
Other Neurons in the Optic Ganglia All the remaining neurons, previously identified light microscopically as Type-I, cluster-forming Type-IV, Type-V, and Type-VI cells, contain small numbers Fig. 7. Portion of E-cell containing small electron-dense elementary granules and lysosome-like dense bodies (LDB). • 9,000 Fig. 8. F-cell with electron-dense elementary granules scattered throughout the cytoplasm. Note rER consisting of slender cisternae; G Golgi complex; LDB lysosome-like dense body x 12,000
518
S. Hisano
Fig. 9. Part of G-cell, showing electron-dense and homogeneous elementary granules. • 18,400 Fig. 10. Granules with coarse matrix of low electron density in G-cell in association with a few "ordinary" granules (arrows). x 21,000
Crustacean Eyestalk Neurosecretory Cells
519
of cored-vesicles. The vesicles, 1,000 A or less in average diameter (Table 1), are characterized by a less dense space around the cores. Most of these vesicles lie near the Golgi complexes (Fig. 13). Golgi cisternae occasionally have an electron-dense content, giving rise to the cored-vesicles (Fig. 13, arrow). The rER consists of cisternae with an electron-lucent lumen. The cells contain lysosome-like dense bodies and multivesicular bodies, which presumably correspond to the AF-positive granules that are noticeable in light micrographs.
Discussion
The elementary granules present in all six types of neurons in the MTGX and in two additional neurons in other parts of the optic ganglia characterize these neurons as peptidergic neurosecretory. Smith (1975) described six neurosecretory cell types in the optic lobe of the crab, Carcinus maenas. It seems that his cell type 5 is comparable to our A- or B-cell, and his cell type 6 to our G-cell, since the granules contained in these cells fall in the same size range and the cells themselves show the same location within the eyestalks (Smith and Naylor, 1972). Empty vesicles similar to those of the H-cell have been reported in neurosecretory cells of teleosts and insects (Fridberg et al., 1966; t3ztan, 1966; Smith and Smith, 1966; Bassurmanova and Panov, 1967; Brady and Maddrell, 1967). Oztan (1966) interpreted them as secretory, because their number varies in relation to photoperiods and seasons. The cored-vesicles in four additional cell types of our material closely resemble in their dimension and appearance the amine-containing vesicles described by Bargmann et al. (1967) and Knowles (1967). Also histochemically, monoaminergic neurons have been demonstrated in the optic ganglia of the crab, Carcinus maenas (Goldstone and Cooke, 1971). Thus, it is highly possible that these four types of cells are aminergic, though it remains to be elucidated whether they are neurosecretory or not. Durand (1956) suggested that his Type 2 cells are the source of the moltinhibiting hormone (MIH) of the crayfish, Orconectes, because the AF-positive droplets of these cells changed in amount in relation to the molting cycle. The cluster-forming Type-IV cells, presumably corresponding to Durand's Type 2 cells, also contain AF-positive material (Hisano, 1974), but electron microscopically these inclusions turned out to be dense bodies rather than neurosecretory granules. Therefore, the hypothesis that these cells are the source of the MIH seems now less feasible. Fig. 11. Electron micrograph of H-cell, containing numerous empty vesicles. Most elementary granules near Golgi complex (G). x 11,500 Fig. 12. Part of H-cell with sER consisting of closely-arranged tubules, x 12,500 Fig, 13. Part of cluster-forming Type-IV cell. Note cored-vesicles (CV) near Golgi complex (G); electron-dense content of Golgi cisternae (arrow). DB dense body; M B multivesicular body. x 17,500
520
S. Hisano
References Bargmann, W.: Neurosecretion. Int. Rev. Cytol. 19, 183-201 (1966) Bargmann, W., Lindner, E., Andres, K.H. : ~ber Synapsen an endokrinen Epithelzellen und die Definition sekretorischer Neurone. Untersuchungen am Zwischenlappen der Katzenhypophyse. Z. Zellforsch. 77, 282-298 (1967) Bassurmanova, O.K., Panov, A.A. : Structure of the neurosecretory system in Lepidoptera. Light and electron microscopy of Type A-neurosecretory cells in the brain of normal and starved larvae of the silkworm Bombyx mori. Gen. comp. Endocr. 9, 245-262 (1967) Bern, H.A. : The properties of neurosecretory cells. Gen. comp. Endocr. , Suppl. 1, 117-132 (1962) Bern, H.A., Knowles, F.G.W. : Neurosecretion. In: Neuroendocrinology, p. 139 186. Eds. L. Martini and W.F. Ganong. New York: Academic Press 1966 Brady, J., Maddrell, S.H.P. : Segmental neurohaemal organs in insects. Z. Zellforsch. 76, 389~,04 (1967) Durand, J.B. : Neurosecretory cell types and their secretory activity in the crayfish. Biol. Bull. 111, 62-76 (1956) Fridberg, G., Bern, H.A., Nishioka, R.S. : The caudal neurosecretory system of the isospondylous teleost, Albula vulpes, from different habitats. Gen. comp. Endocr. 6, 195-212 (1966) Gabe, M. : Neurosecretion. Oxford: Pergamon Press 1966 Goldstone, M.W., Cooke, I.M. : Histochemical localization of monoamines in the crab central nervous system. Z. Zellforsch. 116, 7-19 (1971) Hisano, S. : The eyestalk neurosecretory cell types in the freshwater prawn, Palaemon paucidens. I. A light microscopical study. J. Fac. Sci. Hokkaido Univ., Ser. VI, Zool. 19, 503-514 (1974) Knowles, F.G.W.: Neuronal properties of neurosecretory cells. In: Neurosecretion, p. 8-19. (F. Stutinsky, ed.). Berlin-Heidelberg-New York: Springer 1967 Luft, J.H. :Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409414 (1961) Oztan, N. : The structure of the hypothalamic neurosecretory cells of Zoarces viviparus L. under the conditions of constant dark and light during the reproductive cycle. Z. Zellforsch. 75, 66-82 (1966) Reynolds, E.S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208 212 (1963) Scharrer, B.: Neurohumors and neurohormones: Definitions and terminology. J. neuro-viscer. Relat., Suppl. 9, 1-20 (1969) Smith, G. : The neurosecretory ceils of the optic lobe in Carcinus maenas (L). Cell Tiss. Res. 156, 403-J,09 (1975) Smith, G., Naylor, E. : The neurosecretory system of the eyestalk of Carcinus maenas (Crustacea: Decapoda). J. Zool. (Lond.) 166, 313-321 (1972) Smith, U., Smith, D.S. : Observations on the secretory process in the corpus cardiacum of the stick insect, Carausius morosus L. J. Cell Sci. 1, 59456 (1966)
Received October 21, 1975
Cell and Tissue Research 9 by Springer-Verlag 1976
Neurosecretory Cell Types in the Eyestalk of the Freshwater Prawn Palaemon paucidens An Electron Microscopic Study* Setsuji Hisano Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan
Summary. In the medulla terminalis ganglionic X-organ (MTGX) of the eyestalk of the freshwater prawn, Palaemon paucidens, six peptidergic neurosecretory cell types (A-, B-, C-, D-, E-, and F-cells) are distinguishable on the basis of the different morphology of their elementary granules and rough endoplasmic reticulum (rER). All of these cell types seem to correspond to Type-IIIa cells or dispersing Type-IV cells, that have previously been differentiated at the light microscopic level (Hisano, 1974), as judged from the dimensions of their cell bodies and nuclei. Two other peptidergic neurosecretory cell types that are apparently comparable to the Type-II and Type-IIIb cells (Hisano, 1974), respectively, are recognized in parts of the optic ganglia other than MTGX, and these are now designated as G- and H-cells, respectively. All the remaining cell types, designated as Type-I, cluster-forming Type-IV, Type-V and Type-VI cells in our previous light microscopic study, have small cored-vesicles in their cytoplasm. It remains undecided whether these, possibly aminergic, neurons are neurosecretory or not.
Key words: Neurosecretory cells - Eyestalk - Palaemon - Ultrastrncture. Introduction In a previous study (Hisano, 1974), based on Gomori staining (chrome-hematoxylin phloxine, CHP; aldehyde fuchsin, AF) six types of possibly neurosecretory cells were identified in the optic ganglia of the freshwater prawn, Palaemon paucidens. However, these methods are selective rather than specific for neurosecretory products (Bern, 1962; Bern and Knowles, 1966; Gabe, 1966). Electron microscopy has been used in the present study to arrive at a more reliable Send offprint requests to: Dr. S. Hisano, Zoological Institute, Faculty of Science, Hokkaido Univer-
sity, Sapporo, 060 Japan. * The author wishes to express his sincere appreciation to Prof. T. Aoto for his invaluable advice during the course of this study.
512
S. Hisano
identification. In vertebrates and insects neurosecretory neurons are, as a rule, characterized by electron-dense granules, approximately 500-3,000 A in diameter (Bargmann, 1966; Knowles, 1967; Scharrer, 1969). The same holds for crustaceans.
Material and Methods For electron microscopy only large female Palaemon paucidens, at the intermolt period, were used. Optic ganglia were placed in ice-cold 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) containing 7% sucrose, and fixed in renewed fixative for about 2 hrs at (L4 ~ C. The specimens were postfixed in 1% OsO4 solution in the same buffer for 1-2 hrs at 0 4 ~ C and embedded in Epon 812 (Luft, 1961). Ultra-thin sections were stained with lead citrate (Reynolds, 1963), and examined in a Hitachi HS-7 electron microscope.
Results In the medulla terminalis ganglionic X-organ (MTGX), six types of neurosecretory neurons were identified, based on different features of their elementary granules and rER. These neurons seem to correspond to the cells previously identified light microscopically as Type-IIIa or as dispersing Type-IV cells (Hisano, 1974), as judged from the dimensions of the cell bodies and nuclei. These six cell types will now be referred to as A-, B-, C-, D-, E-, and F-cells, respectively (Table 1).
Table 1. Interrelation between neurosecretory cell types classified in the present and the previous studies Present study
Size of elementary granules or vesicles
Hisano (1974)
Remarks
Type-Ilia cell or Dispersing Type-IV cell
Peptidergic
(A) Elementary granules A-cell B-cell C-cell D-cell E-ceU F-cell
1,000~,300 1,000-2,100 1,000-1,700 700 1,400 700-1,300 700-1,300
G-cell
700-2,800
Type-II cell
Peptidergic
H-cell
800-1,200
Type-IIIb cell
Peptidergic
Type-I cell Cluster-forming Type-IV cell Type-V cell Type-VI cell
Aminergic (.9)
Vesicles 900-1,400 600-1,000 700-1,200 500- 900 a
(1,600)" (1,500) (1,300) (1,000) (1,000) (1,000)
(1,000) a (900) (900) (700)
Mean diameter shown in parentheses.
Crustacean Eyestalk Neurosecretory Cells
513
30-
J,J
,,=, 20,,=
',,,
i i
', ', ,.,..
==,
r
10-
0b0 1 a'oo GRANULEDIAMETER(,~)
2 o'oo
Fig. 1. Frequency distribution of diameters of elementary granules in six neurosecretory cell types of the M T G X . o - - o F-cell
A-cell; n - - i
B-cell; o - - - o
C-cell; o - - - o
D-cell; 9 . . . . . 9 E - c e l l ; o - - o
A - Cell
The not very abundant elementary granules of the A-cell are spheroidal and rather large (1,000-2,300 ,~; 1,600 A average, in diameter; Figs. 1, 2). A highly electron-dense matrix, homogeneous in appearance, is bounded by a thin limiting membrane. Most granules are dispersed throughout the cytoplasm. In the well developed Golgi complex the cisternae are occasionally enlarged at the ends, and contain a moderately dense material, apparently giving rise to the elementary granules. The rER consists of flattened cisternae running parallel to each other. The intra-cisternal space is slightly electron-dense owing to the presence of some flocculent material.
B-Cell
A relatively large number of elementary granules are present. The Golgi complex frequently contains electron-dense material in its cisternae (Fig. 3). The elementary granules, 1,000-2,100 A (average 1,500 A; Fig. 1), are round. Their electron density is lower than that of those in the A-cells. The rER consists of a number of short cisternae and vesicles with an electron-lucent and relatively wide lumen. Lysosome-like dense bodies of various sizes and shapes often contain a few
514
S. Hisano
Crustacean Eyestalk Neurosecretory Cells
515
elementary granules (Fig. 3, arrow). In a few cells with a number of semicircular subunits, there are large inclusions, 4-5 la in diameter (Fig. 4). The exact nature and functional significance, if any, of these inclusions are still unknown.
C-Ce// This cell has round or spheroidal medium-sized electron-dense elementary granules, approximately 1,000-1,700,~ (average 1,300A; Figs. 1, 5). Their number fluctuates considerably in relation to the morphology of the rER. In cells with cisternal or vesicular rER a large number of elementary granules are present, whereas in cells whose rER consists of distended sacs, only a few elementary granules are observed near the Golgi elements. These differences suggest different phases in the secretory cycle of the C-cells.
D-Ce// In the D-cell, the small elementary granules, approximately 700-1,400 A (average 1,000 A), are round (Figs. 1, 6). The cisternae of the rER have an electron-lucent, wide lumen. Golgi complexes are well developed, with often dense contents (Fig. 6, arrow).
E-Ce// In this cell, a small number of round elementary granules, 700-1,300 A (average 1,000 A) are present (Figs. 1, 7). The relatively abundant rER consists of slender parallel cisternae. The cytoplasm between cisternae is slightly more electrondense than the remaining cytoplasm (Fig. 7), due to the presence of flocculent material. Also found here and there in the cytoplasm are vacuoles of varying size and shape, and glycogen particles. The vacuoles always contain an anastomosing filamentous material whose functional role is unknown.
F-Ce// The elementary granules of this cell are round and small, measuring approximately 700-1,300 A (average 1,000 A; Fig. 1). They are electron-dense and scattered among the other organelles (Fig. 8). The content of the slender rER cister-
Fig. 2. Electron micrograph of A-cell containing highly electron-dense elementary granules. Note rER consisting of parallel cisternae and lysosome-like bodies (Ly). • 18,000 Fig. 3. B-cell with many elementary granules of moderate electron density. Note lysosome-like dense body (arrow) containing several elementary granules, x 20,700 Fig.4. Large inclusion body filled with numerous semicircular subunits in B-cell. x 9,100
516
S. Hisano
Fig. 5. C-cell with cisternal and vesicular rER. Elementary granules actively elaborated in Golgi complex (G). x 18,400 Fig. 6. Electron micrograph of D-cell. Note electron-dense content (arrow) in Golgi complex (G). • 16,100
Crustacean Eyestalk Neurosecretory Cells
517
nae is moderately electron-dense. Their distribution in the cytoplasm is disorderly.
Neurosecretory Cells of the Optic Ganglia other than the MTGX At least two additional types of neurons, located in the optic ganglia other than the MTGX, were found to be neurosecretory. These are now termed G-cell and H-cell; they were previously called Type-II and Type-IIIb (Hisano, 1974; see Table 1). G-Ce// The G-cell contains clumps of numerous elementary granules throughout the cytoplasm (Fig. 9), which seem to correspond to the masses of "Gomori-positive" material in light micrographs. The granules are round or spheroidal and range from 700 to 2,800 A. Most granules appear "homogeneous". Some show a coarse matrix of low electron density (Fig. 10, arrows). The usual limiting membrane of these granules is occasionally torn. Electron-dense material is always present in Golgi cisternae. The lamellar rER, for the most part, has an electron-lucent content. In some cells with coarse elementary granules of low electron density the intracisternal material is a little more dense. H-Ce// Elementary granules, approximately 800-1,200 A, are very scarce, although this cell contains abundant "Gomori-positive" material (Hisano, 1974). Most of the electron-dense granules lie near the Golgi complexes (Fig. 11). A large number of empty vesicles occur in the perinuclear region and at the cell periphery. These vesicles are round or oblong, varying from 700-1,900 A (average 1,300 .~). There still remains much conjecture as to the origin of these vesicles. Both rER and sER occur in this cell. The rER has the form of well-developed lamellae, and the sER is composed of closely-arranged tubules of fairly uniform width (Fig. 12). The role of the latter is unclear.
Other Neurons in the Optic Ganglia All the remaining neurons, previously identified light microscopically as Type-I, cluster-forming Type-IV, Type-V, and Type-VI cells, contain small numbers Fig. 7. Portion of E-cell containing small electron-dense elementary granules and lysosome-like dense bodies (LDB). • 9,000 Fig. 8. F-cell with electron-dense elementary granules scattered throughout the cytoplasm. Note rER consisting of slender cisternae; G Golgi complex; LDB lysosome-like dense body x 12,000
518
S. Hisano
Fig. 9. Part of G-cell, showing electron-dense and homogeneous elementary granules. • 18,400 Fig. 10. Granules with coarse matrix of low electron density in G-cell in association with a few "ordinary" granules (arrows). x 21,000
Crustacean Eyestalk Neurosecretory Cells
519
of cored-vesicles. The vesicles, 1,000 A or less in average diameter (Table 1), are characterized by a less dense space around the cores. Most of these vesicles lie near the Golgi complexes (Fig. 13). Golgi cisternae occasionally have an electron-dense content, giving rise to the cored-vesicles (Fig. 13, arrow). The rER consists of cisternae with an electron-lucent lumen. The cells contain lysosome-like dense bodies and multivesicular bodies, which presumably correspond to the AF-positive granules that are noticeable in light micrographs.
Discussion
The elementary granules present in all six types of neurons in the MTGX and in two additional neurons in other parts of the optic ganglia characterize these neurons as peptidergic neurosecretory. Smith (1975) described six neurosecretory cell types in the optic lobe of the crab, Carcinus maenas. It seems that his cell type 5 is comparable to our A- or B-cell, and his cell type 6 to our G-cell, since the granules contained in these cells fall in the same size range and the cells themselves show the same location within the eyestalks (Smith and Naylor, 1972). Empty vesicles similar to those of the H-cell have been reported in neurosecretory cells of teleosts and insects (Fridberg et al., 1966; t3ztan, 1966; Smith and Smith, 1966; Bassurmanova and Panov, 1967; Brady and Maddrell, 1967). Oztan (1966) interpreted them as secretory, because their number varies in relation to photoperiods and seasons. The cored-vesicles in four additional cell types of our material closely resemble in their dimension and appearance the amine-containing vesicles described by Bargmann et al. (1967) and Knowles (1967). Also histochemically, monoaminergic neurons have been demonstrated in the optic ganglia of the crab, Carcinus maenas (Goldstone and Cooke, 1971). Thus, it is highly possible that these four types of cells are aminergic, though it remains to be elucidated whether they are neurosecretory or not. Durand (1956) suggested that his Type 2 cells are the source of the moltinhibiting hormone (MIH) of the crayfish, Orconectes, because the AF-positive droplets of these cells changed in amount in relation to the molting cycle. The cluster-forming Type-IV cells, presumably corresponding to Durand's Type 2 cells, also contain AF-positive material (Hisano, 1974), but electron microscopically these inclusions turned out to be dense bodies rather than neurosecretory granules. Therefore, the hypothesis that these cells are the source of the MIH seems now less feasible. Fig. 11. Electron micrograph of H-cell, containing numerous empty vesicles. Most elementary granules near Golgi complex (G). x 11,500 Fig. 12. Part of H-cell with sER consisting of closely-arranged tubules, x 12,500 Fig, 13. Part of cluster-forming Type-IV cell. Note cored-vesicles (CV) near Golgi complex (G); electron-dense content of Golgi cisternae (arrow). DB dense body; M B multivesicular body. x 17,500
520
S. Hisano
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Received October 21, 1975
