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Association of elementary neurosecretory granules with the Golgi complex
J. ULTRASTRUCTURERESEARCH 5, 311-320 (1961)
311
Association of Elementary Neurosecretory Granules with the Golgi Complex ~ HOWARD A. BERN, RICHARD S. NISHIOKA and IRVINE R. HAGADORN
Department of Zoology and its Cancer Research Genetics Laboratory, University of California, Berkeley Received February 6, 1961 Electron microscope observations have been made on neurosecretory cells from the supraesophageal ganglion of the leech Theromyzon rude, from the pars intercerebralis of the cockroach Periplaneta americana, and from the preoptic nucleus of the hypothalamus of the frog Rana pipiens. A consistent association was noted between Golgi membranes and elementary neurosecretory granules. The Golgi apparatus was generally more prominent in the invertebrate neurons than in the amphibian neurons. There is a scarcity of information available on the fine structure of the neurosecretory perikaryon. Fingerman and Aoto (7) have published a few indistinct micrographs of crustacean neurosecretory cell bodies, and Miyawaki (14) has made some tentative suggestions about the relationship of various cell organelles to neurosecretion in brachyuran crustaceans, based in part on electron microscope observations. Some details have been provided on the ultrastructure of the Dahlgren cells of the teleost caudal neurosecretory system (6, 18), and a neuron of the nucleus tuberis is included in a study of the rabbit hypothalamus (9). Most recently, as this paper was being completed, a thorough study of the preoptic nucleus of the goldfish by Palay (16) appeared, which presented a picture similar to the findings reported herein, and Nishiitsutsuji-uwo (15) has noted her findings on lepidopteran neurosecretory cells. Unlike the neurosecretory nuclei, neurohemal organs and neurosecretory tracts have received considerable attention. In all areas studied (neurohypophysis and urohypophysis of vertebrates; corpus cardiacum and nervus corporis allati of insects; sinus gland, pericardial organs, and post-commissural organs of crustaceans--cf. 2), the stainable neurosecretory material in axons and in axonal bulbs is seen to be composed of elementary neurosecretory granules, in the 1000-3000-A size range, which are often present in two size classes within this range. The generally accepted Scharrer-Bargmann concept of neurosecretion postulates synthesis of the neurosecrei Aided by Grant G-8805 from the National Science Foundation. 21 -- 61173319 J . Ultrastrueture Research
312
HOWARD A. BERN ET AL.
tory material in the perikaryon and its distal transport to the point of storage and ultimate release from the axon termination. Accordingly, special interest exists as to the exact locus of formation of the neurosecretory granules in the cell body. The present note illustrates a consistent association of elementary neurosecretory granules with well-developed Golgi complexes in three varieties of neurosecretory perikarya: from the supraesophageal ganglion of the rhynchobdellid leech, T. rude (8); from the pars intercerebralis of the brain of the cockroach, P. americana; and from the preoptic nucleus of the hypothalamus of the frog, R. pipiens,
MATERIALS AND METHODS The supraesophageal ganglia of 5 adult leeches (T. rude) were fixed in 1% osmium tetroxide buffered at pH 7.5 with veronal-acetate to which was added 0.116 g/ml of sucrose (3). The pars intercerebralis regions of the brains of 3 mature female cockroaches (P. americana) were immersed in the fixative of Sano and Knoop (18), to which was added sucrose (0.03 g/ml) instead of the salt solution. The hypothalamic regions of the brains of 7 adult male frogs (R. pipiens) were fixed in l% osmium tetroxide buffered with 0.2 M collidine (1). All of the tissues were fixed at 0-5°C for 2-3 hours, rinsed with distilled water, dehydrated in a graded series of alcohols, infiltrated with n-butyl methacrylate, and polymerized at 60°C with the catalyst 2,4-dichlorobenzoyl peroxide. Sections were cut with glass knives on the Porter-Blum microtome. The sections were picked up on Formvar-coated copper grids and examined in a RCA EMU 3 E electron microscope.
OBSERVATIONS The emphasis in the present communication is specifically upon the relationship between elementary neurosecretory granules and the Golgi apparatus; other details of perikaryon structure are being reserved for a later paper. Within the cytoplasm of the neurosecretory cell bodies are seen variable numbers of homogeneously dense structures, which fall in the size range characteristic of elementary neurosecretory granules (1500-2500 A). Many of the invertebrate secretory neurons studied showed particularly prominent Golgi complexes consisting of closely stacked, agranular paired membranes, arranged in parallel (Figs. l-4). Each membrane in profile was about 60 • thick, and the
Ft~. 1. Neurosecretory perikaryon of the leech supraesophageal ganglion, containing three prominent Golgi complexes (g) near the nucleus (n). Scattered throughout the cytoplasm are numerous membranes of the endoplasmic reticulum (r). x 8000. FIG. 2. Neurosecretory perikaryon of the cockroach pars intercerebralis, with several Golgi complexes (g). The cell body is surrounded by extensive glial membranes (s). Note the large round inclusion bodies (i). × 4000.
314
HOWARD A. BERN ET AL.
members of a pair were separated from each other by approximately 50 A. The intramembranal spaces were slightly denser than the intermembranal spaces. Groups of these membranes were encountered in the form of curvilinear (Fig. 5) to horseshoeshaped bodies (Figs. 3-4), often in the proximity of the nucleus. Simple short segments of membrane systems which could have been parts of a more complex system were commonly encountered. Associated with the groups of closely stacked membranes were other paired membranes with intermittent constrictions and swellings. The latter membranes usually were separated from the uniform membranes by more than 50 ~ . Peripheral to these membranes were vesicles of variable density and size. Smaller vesicles (300-600 A in diameter) were aggregated close to the Golgi membranes and larger vesicles with highly osmiophilic cores, measuring up to 1500 A, often were located at some distance. Many cells in the leech contained enormous numbers of elementary granules. In these cells only a trace of the Golgi apparatus was noted. On the other hand, cells with sparsely distributed granules showed massive Golgi systems. It is possible that the former cells were storing secretory material, whereas the latter were at the beginning of a secretory cycle, engaged in active synthesis of the secretory product. The preoptic neurons of the frog (Fig. 6) had less extensive Golgi areas than the invertebrate neurons; however, the relation of electron-dense granules to the Golgi membranes was indicated clearly here also. Another notable difference between invertebrate and vertebrate cells was the presence of large, sac-like dilatations in association with Golgi membranes in the latter. It is conceivable that these sacs were a result of fixation in a hypotonic solution.
DISCUSSION To our knowledge, these represent the first published ultrastructural studies of neurosecretory cell bodies from the Annelida. 1 Among the Insecta, Schultz (20) has stated that cells of the pars intercerebralis of the moth Celerio lineata show a high 1 Herlant-Meewis and her associates have examined neurosecretory cells of various invertebrates (personal communication), and Scharrer and Brown (19) have completed an elegant study of neurosecretion in the brain of Lurnbricus terresn'is.
Flo. 3. High magnification of two Golgi complexes (g) from the leech. Stages in granule transformation are evident. The large elementary neurosecretory granules (e) measure 1800-2000 ~. n, nucleus. x 23,000. FIG. 4. High magnification of two Golgi complexes (g) from the cockroach. Several stages in the transformation of elementary neurosecretory granules (e) are illustrated. The largest granules measure about 1500 ~ in diameter. At the top is a glial process (p) projecting into the cell body. x 33,000.
FIG. 5. H i g h magnification of two curvilinear Golgi a p p a r a t u s e s (g) f r o m the cockroach. T h e t r a n s f o r m a t i o n from small e m p t y vesicles (v) to highly osmiophilic elementary granules (el) , is suggested. A vesicle with a dense core is s h o w n attached to Golgi m e m b r a n e s at e2. rn, m i t o c h o n d r i o n , x 45,000.
FIG. 6. Part of a neurosecretory cell body from the frog preoptic nucleus. Golgi complex (g) contains some very large clear vesicles (v). The large elementary granules measure about 1500/~, and some appear to be included in dilatatians of the Golgi membranes (e). n, nucleus; m, mitochondrion, x 33,000.
318
H O W A R D A. BERN ET AL.
concentration of elementary granules (1000-3000 A), and Nishiitsutsuji-uwo (15) has reported on the fine structure of pars intercerebralis cells of three additional lepidopteran species. Our present focus on the Golgi system is part of a more general study of the fine structure of neurosecretory cells and of the neurosecretory process being undertaken in our laboratory. Neurocytologists have concerned themselves for some time with the various inclusion bodies demonstrable in neurons and suggestive of secretory activity. In particular, gastropod neurons, 1 the Purkinje cells of the cerebellum, and vertebrate sympathetic neurons have received considerable study. In our material large organellar systems consisting of membranes and vesicles were found regularly in cells from areas considered by students of neurosecretion as being unquestionably secretory. The organelles described herein, presumably equivalent to the Golgi apparatus of other synthetically active cells, are associated with granules that can be visualized in a series of increasing size and electron density to suggest their formation in the Golgi apparatus itself. These granules, once formed, are often contained within limiting membranes, many of which appear to be pinched off the ends of the paired Golgi membranes. As with instances of the association of protein material with the Golgi apparatus (e.g., melanin formation--4, 22; milk protein formation--21), it cannot be concluded that the basic neurosecretory material is synthesized by the Golgi vesicles; however, the supramolecular organization of the elementary granule is possibly accomplished in the area of the Golgi apparatus and with its apparent intervention. The entry of formed neurosecretory granules into the Golgi membrane system for ultimate discharge through the cell surface represents an alternative possibility. However, the formation of tiny vesicles at the ends of elongate sacs, which progressively develop electron-dense centers, is suggested strongly by the electron microscope evidence. In addition, the accepted locus for secretion discharge in most neurosecretory cells is at a relatively great distance from the cell b o d y - - a t the end of an axon that may proceed many millimeters from the neurosecretory nucleus to the neurohemal organ. In the axon we have been unable to find evidence of elongate sacs or membrane systems that might be connected with the Golgi centers. Canals of the neurofibrillar system recently described by Wigglesworth (23) as being linked up with the Golgi bodies in ordinary neurons of the cockroach were not apparent in our electron micrographs of pars intercerebralis cells of the same species, nor in those of the leech cells. The leech cells, furthermore, showed densely packed fibrillar strands in the perikaryon, which appeared to reflect the intensely argyrophilic neurofibrillar 1 In a recent electron microscope study, Dalton (5) has commented on the relationship of presumed neurosecretory granules to the Golgi complex in neurons of the dorsal ganglion of the snail, Helix pomatia.
NEUROSECRETORY CELLS AND GOLGI COMPLEX
319
network seen in the light microscope (8), and which evidenced no relationship with the Golgi systems. Parameswaran (17) and Matsumoto (10) have presented evidence for the discharge of secretion from the perikaryon itself by some neurosecretory cells in the fused thoracic ganglion of crabs. If this situation obtained in the leech, for example, or if a canalicular system such as that described by Wigglesworth (23) were present, the inverse relationship between the extent of the Golgi apparatus and the number of neurosecretory granules would be readily explicable: the more extensive the Golgi apparatus, the more efficient the discharge of secretory product. Nishiitsutsuji-uwo (15) stated that the elementary neurosecretory granules in lepidopteran neurosecretory cells arose from constriction and fragmentation of mitochondria and that they also received some material from the Golgi apparatus. We found no evidence in the cockroach, nor in other neurosecretory cells, of any relationship of this sort between mitochondria and the elementary granules. Miyawaki (12-14) has reported recently on the cytology of various neurosecretory cells in decapod crustaceans. The large horseshoe-shaped inclusion bodies in the crab Gaetice depressus (see especially Fig. 18 in 12) are probably identical to the structures we have described herein. Miyawaki's electron micrograph (Fig. 5, 14) is strongly suggestive in this respect. Unfortunately, the identities of lipochondria, neurosecretory globules, Nissl bodies, Golgi apparatus, and mitochondria are largely lost in Miyawaki's discussion. Maynard (11) also has commented on "laminar profiles" involved in the synthesis of neurosecretory material in the C-cells of the thoracic ganglia of several brachyurans. Data in support of the origin of neurosecretory granules in the Golgi apparatus have been presented by Sano and Knoop (18) from studies on the Dahlgren (caudal neurosecretory) cells of the tench, Tinca vulgaris. These cells contain a "Gomorinegative" secretory material that appears to arise in much the same manner described herein for annelid, insect, and amphibian cells. We have examined the caudal neurosecretory cells of three other teleost species: the mullet, Mugil cephalus; the goldfish Carassius auratus; and Tilapia mossambica. In all instances, we can find a degree of association between formed granules and Golgi vesicles that is suggestive of a relationship. However, neither Sano and Knoop nor we have encountered the prominent organellar complexes reported here for other neurosecretory neurons. Until the appearance of the thorough study of the preoptic nucleus of the goldfish C. auratus by Palay (16), no information was available on the fine structure of hypothalamic neurosecretory perikarya. Our observations on the preoptic nucleus of Rana are in accord with those of Palay in regard to the association of the typical elementary neurosecretory granules (in the 1000-1500-~ size range) with Golgi membranes. Of interest is the fact that the Golgi complex is considerably less prominent in verte-
320
HOWARD A. BERN ET AL.
brate as compared with invertebrate neurosecretory perikarya. Both the Dahlgren cells and the preoptic neurons of vertebrates show the same association between membranes and granules as do the invertebrate neurons, but the m e m b r a n e system is generally more diffuse in the former. We are indebted to Dr. Dorothy Pitelka for suggestions and for discussion of this material.
REFERENCES 1. BENNETT, H. S. and LUFT, J. H., J. Biophys. Biochem. Cytol. 6, 113 (1959). 2. BERN, H. A. and HAGADORN, I. R., in BULLOCK, T. H. and HORRIDGE, G. A. The Nervous System in Invertebrates. Freeman, San Francisco, in press. 3. CAULFIELD,J. B., J. Biophys. Biochem. Cytol. 3, 827 (1957). 4. DALTON, A. J., Lab. Invest. 8, 510 (1959). 5. - in M. D. Anderson Hospital and Tumor Institute, Cell Physiology of Neoplasia, p. 161-184. University of Texas Press, Austin, 1960. 6. ENAMI, M. and IMAI, K., Proe. Japan Acad. 34, 164 (1958). 7. F~NGERMAN, M. and AOTO, T., Trans. Am. Microscop. Soc. 78, 305 (1959). 8. HAGADORN,I. R., J. Morphol. 102, 55 (1958). 9. L6BLICH, H. J. and KNEZ~VlC, M., Beitriigepathol. Anat. u. allgem. Pathol. 122, 1 (1960). 10. MATSUMOTO,K., Biol. J. Okayama Univ. 4, 103 (1958). 11. MAYNARD, D. M., Gen. Comp. Endocrinol., in press (1961). 12. MIYAWAKI, M., Kumamoto J. Sci. Ser. B., See. 2, 5, 1 (1960). 13. - - - - ibid. 5, 21 (1960). 14. - ibid. 5, 29 (1960). 15. NISHnTSUTSUJI-Uwo, J., Nature 183, 953 (1960). 16. PALAY, S. L., Anat. Record 138, 417 (1960). 17. PARAMESWARAN,R., Quart. d. Microscop. Sci. 97, 75 (1956). 18. SANO, Y. and KNOOt', A., Z. Zellforsch. u. mikroskop. Anat. 49, 464 (1959). 19. SCHARRER,E. and BROWN, S., Anat. Record 139, 271 (1961). 20. SCHULTZ,R. L., J. Ultrastructure Research 3, 320 (1960). 21. WELLINGS,S. R. and DE OME, K. B., J. Biophys. Biochem. Cytol. 9, 479 (1961). 22. WELLINGS, S. R. and SIEGEL, B. V., J. Ultrastructure Research 3, 147 (1959). 23. WIGGLFSWORTH,V. B., Quart. d. Microscop. Sci. 101, 391 (1960).
311
Association of Elementary Neurosecretory Granules with the Golgi Complex ~ HOWARD A. BERN, RICHARD S. NISHIOKA and IRVINE R. HAGADORN
Department of Zoology and its Cancer Research Genetics Laboratory, University of California, Berkeley Received February 6, 1961 Electron microscope observations have been made on neurosecretory cells from the supraesophageal ganglion of the leech Theromyzon rude, from the pars intercerebralis of the cockroach Periplaneta americana, and from the preoptic nucleus of the hypothalamus of the frog Rana pipiens. A consistent association was noted between Golgi membranes and elementary neurosecretory granules. The Golgi apparatus was generally more prominent in the invertebrate neurons than in the amphibian neurons. There is a scarcity of information available on the fine structure of the neurosecretory perikaryon. Fingerman and Aoto (7) have published a few indistinct micrographs of crustacean neurosecretory cell bodies, and Miyawaki (14) has made some tentative suggestions about the relationship of various cell organelles to neurosecretion in brachyuran crustaceans, based in part on electron microscope observations. Some details have been provided on the ultrastructure of the Dahlgren cells of the teleost caudal neurosecretory system (6, 18), and a neuron of the nucleus tuberis is included in a study of the rabbit hypothalamus (9). Most recently, as this paper was being completed, a thorough study of the preoptic nucleus of the goldfish by Palay (16) appeared, which presented a picture similar to the findings reported herein, and Nishiitsutsuji-uwo (15) has noted her findings on lepidopteran neurosecretory cells. Unlike the neurosecretory nuclei, neurohemal organs and neurosecretory tracts have received considerable attention. In all areas studied (neurohypophysis and urohypophysis of vertebrates; corpus cardiacum and nervus corporis allati of insects; sinus gland, pericardial organs, and post-commissural organs of crustaceans--cf. 2), the stainable neurosecretory material in axons and in axonal bulbs is seen to be composed of elementary neurosecretory granules, in the 1000-3000-A size range, which are often present in two size classes within this range. The generally accepted Scharrer-Bargmann concept of neurosecretion postulates synthesis of the neurosecrei Aided by Grant G-8805 from the National Science Foundation. 21 -- 61173319 J . Ultrastrueture Research
312
HOWARD A. BERN ET AL.
tory material in the perikaryon and its distal transport to the point of storage and ultimate release from the axon termination. Accordingly, special interest exists as to the exact locus of formation of the neurosecretory granules in the cell body. The present note illustrates a consistent association of elementary neurosecretory granules with well-developed Golgi complexes in three varieties of neurosecretory perikarya: from the supraesophageal ganglion of the rhynchobdellid leech, T. rude (8); from the pars intercerebralis of the brain of the cockroach, P. americana; and from the preoptic nucleus of the hypothalamus of the frog, R. pipiens,
MATERIALS AND METHODS The supraesophageal ganglia of 5 adult leeches (T. rude) were fixed in 1% osmium tetroxide buffered at pH 7.5 with veronal-acetate to which was added 0.116 g/ml of sucrose (3). The pars intercerebralis regions of the brains of 3 mature female cockroaches (P. americana) were immersed in the fixative of Sano and Knoop (18), to which was added sucrose (0.03 g/ml) instead of the salt solution. The hypothalamic regions of the brains of 7 adult male frogs (R. pipiens) were fixed in l% osmium tetroxide buffered with 0.2 M collidine (1). All of the tissues were fixed at 0-5°C for 2-3 hours, rinsed with distilled water, dehydrated in a graded series of alcohols, infiltrated with n-butyl methacrylate, and polymerized at 60°C with the catalyst 2,4-dichlorobenzoyl peroxide. Sections were cut with glass knives on the Porter-Blum microtome. The sections were picked up on Formvar-coated copper grids and examined in a RCA EMU 3 E electron microscope.
OBSERVATIONS The emphasis in the present communication is specifically upon the relationship between elementary neurosecretory granules and the Golgi apparatus; other details of perikaryon structure are being reserved for a later paper. Within the cytoplasm of the neurosecretory cell bodies are seen variable numbers of homogeneously dense structures, which fall in the size range characteristic of elementary neurosecretory granules (1500-2500 A). Many of the invertebrate secretory neurons studied showed particularly prominent Golgi complexes consisting of closely stacked, agranular paired membranes, arranged in parallel (Figs. l-4). Each membrane in profile was about 60 • thick, and the
Ft~. 1. Neurosecretory perikaryon of the leech supraesophageal ganglion, containing three prominent Golgi complexes (g) near the nucleus (n). Scattered throughout the cytoplasm are numerous membranes of the endoplasmic reticulum (r). x 8000. FIG. 2. Neurosecretory perikaryon of the cockroach pars intercerebralis, with several Golgi complexes (g). The cell body is surrounded by extensive glial membranes (s). Note the large round inclusion bodies (i). × 4000.
314
HOWARD A. BERN ET AL.
members of a pair were separated from each other by approximately 50 A. The intramembranal spaces were slightly denser than the intermembranal spaces. Groups of these membranes were encountered in the form of curvilinear (Fig. 5) to horseshoeshaped bodies (Figs. 3-4), often in the proximity of the nucleus. Simple short segments of membrane systems which could have been parts of a more complex system were commonly encountered. Associated with the groups of closely stacked membranes were other paired membranes with intermittent constrictions and swellings. The latter membranes usually were separated from the uniform membranes by more than 50 ~ . Peripheral to these membranes were vesicles of variable density and size. Smaller vesicles (300-600 A in diameter) were aggregated close to the Golgi membranes and larger vesicles with highly osmiophilic cores, measuring up to 1500 A, often were located at some distance. Many cells in the leech contained enormous numbers of elementary granules. In these cells only a trace of the Golgi apparatus was noted. On the other hand, cells with sparsely distributed granules showed massive Golgi systems. It is possible that the former cells were storing secretory material, whereas the latter were at the beginning of a secretory cycle, engaged in active synthesis of the secretory product. The preoptic neurons of the frog (Fig. 6) had less extensive Golgi areas than the invertebrate neurons; however, the relation of electron-dense granules to the Golgi membranes was indicated clearly here also. Another notable difference between invertebrate and vertebrate cells was the presence of large, sac-like dilatations in association with Golgi membranes in the latter. It is conceivable that these sacs were a result of fixation in a hypotonic solution.
DISCUSSION To our knowledge, these represent the first published ultrastructural studies of neurosecretory cell bodies from the Annelida. 1 Among the Insecta, Schultz (20) has stated that cells of the pars intercerebralis of the moth Celerio lineata show a high 1 Herlant-Meewis and her associates have examined neurosecretory cells of various invertebrates (personal communication), and Scharrer and Brown (19) have completed an elegant study of neurosecretion in the brain of Lurnbricus terresn'is.
Flo. 3. High magnification of two Golgi complexes (g) from the leech. Stages in granule transformation are evident. The large elementary neurosecretory granules (e) measure 1800-2000 ~. n, nucleus. x 23,000. FIG. 4. High magnification of two Golgi complexes (g) from the cockroach. Several stages in the transformation of elementary neurosecretory granules (e) are illustrated. The largest granules measure about 1500 ~ in diameter. At the top is a glial process (p) projecting into the cell body. x 33,000.
FIG. 5. H i g h magnification of two curvilinear Golgi a p p a r a t u s e s (g) f r o m the cockroach. T h e t r a n s f o r m a t i o n from small e m p t y vesicles (v) to highly osmiophilic elementary granules (el) , is suggested. A vesicle with a dense core is s h o w n attached to Golgi m e m b r a n e s at e2. rn, m i t o c h o n d r i o n , x 45,000.
FIG. 6. Part of a neurosecretory cell body from the frog preoptic nucleus. Golgi complex (g) contains some very large clear vesicles (v). The large elementary granules measure about 1500/~, and some appear to be included in dilatatians of the Golgi membranes (e). n, nucleus; m, mitochondrion, x 33,000.
318
H O W A R D A. BERN ET AL.
concentration of elementary granules (1000-3000 A), and Nishiitsutsuji-uwo (15) has reported on the fine structure of pars intercerebralis cells of three additional lepidopteran species. Our present focus on the Golgi system is part of a more general study of the fine structure of neurosecretory cells and of the neurosecretory process being undertaken in our laboratory. Neurocytologists have concerned themselves for some time with the various inclusion bodies demonstrable in neurons and suggestive of secretory activity. In particular, gastropod neurons, 1 the Purkinje cells of the cerebellum, and vertebrate sympathetic neurons have received considerable study. In our material large organellar systems consisting of membranes and vesicles were found regularly in cells from areas considered by students of neurosecretion as being unquestionably secretory. The organelles described herein, presumably equivalent to the Golgi apparatus of other synthetically active cells, are associated with granules that can be visualized in a series of increasing size and electron density to suggest their formation in the Golgi apparatus itself. These granules, once formed, are often contained within limiting membranes, many of which appear to be pinched off the ends of the paired Golgi membranes. As with instances of the association of protein material with the Golgi apparatus (e.g., melanin formation--4, 22; milk protein formation--21), it cannot be concluded that the basic neurosecretory material is synthesized by the Golgi vesicles; however, the supramolecular organization of the elementary granule is possibly accomplished in the area of the Golgi apparatus and with its apparent intervention. The entry of formed neurosecretory granules into the Golgi membrane system for ultimate discharge through the cell surface represents an alternative possibility. However, the formation of tiny vesicles at the ends of elongate sacs, which progressively develop electron-dense centers, is suggested strongly by the electron microscope evidence. In addition, the accepted locus for secretion discharge in most neurosecretory cells is at a relatively great distance from the cell b o d y - - a t the end of an axon that may proceed many millimeters from the neurosecretory nucleus to the neurohemal organ. In the axon we have been unable to find evidence of elongate sacs or membrane systems that might be connected with the Golgi centers. Canals of the neurofibrillar system recently described by Wigglesworth (23) as being linked up with the Golgi bodies in ordinary neurons of the cockroach were not apparent in our electron micrographs of pars intercerebralis cells of the same species, nor in those of the leech cells. The leech cells, furthermore, showed densely packed fibrillar strands in the perikaryon, which appeared to reflect the intensely argyrophilic neurofibrillar 1 In a recent electron microscope study, Dalton (5) has commented on the relationship of presumed neurosecretory granules to the Golgi complex in neurons of the dorsal ganglion of the snail, Helix pomatia.
NEUROSECRETORY CELLS AND GOLGI COMPLEX
319
network seen in the light microscope (8), and which evidenced no relationship with the Golgi systems. Parameswaran (17) and Matsumoto (10) have presented evidence for the discharge of secretion from the perikaryon itself by some neurosecretory cells in the fused thoracic ganglion of crabs. If this situation obtained in the leech, for example, or if a canalicular system such as that described by Wigglesworth (23) were present, the inverse relationship between the extent of the Golgi apparatus and the number of neurosecretory granules would be readily explicable: the more extensive the Golgi apparatus, the more efficient the discharge of secretory product. Nishiitsutsuji-uwo (15) stated that the elementary neurosecretory granules in lepidopteran neurosecretory cells arose from constriction and fragmentation of mitochondria and that they also received some material from the Golgi apparatus. We found no evidence in the cockroach, nor in other neurosecretory cells, of any relationship of this sort between mitochondria and the elementary granules. Miyawaki (12-14) has reported recently on the cytology of various neurosecretory cells in decapod crustaceans. The large horseshoe-shaped inclusion bodies in the crab Gaetice depressus (see especially Fig. 18 in 12) are probably identical to the structures we have described herein. Miyawaki's electron micrograph (Fig. 5, 14) is strongly suggestive in this respect. Unfortunately, the identities of lipochondria, neurosecretory globules, Nissl bodies, Golgi apparatus, and mitochondria are largely lost in Miyawaki's discussion. Maynard (11) also has commented on "laminar profiles" involved in the synthesis of neurosecretory material in the C-cells of the thoracic ganglia of several brachyurans. Data in support of the origin of neurosecretory granules in the Golgi apparatus have been presented by Sano and Knoop (18) from studies on the Dahlgren (caudal neurosecretory) cells of the tench, Tinca vulgaris. These cells contain a "Gomorinegative" secretory material that appears to arise in much the same manner described herein for annelid, insect, and amphibian cells. We have examined the caudal neurosecretory cells of three other teleost species: the mullet, Mugil cephalus; the goldfish Carassius auratus; and Tilapia mossambica. In all instances, we can find a degree of association between formed granules and Golgi vesicles that is suggestive of a relationship. However, neither Sano and Knoop nor we have encountered the prominent organellar complexes reported here for other neurosecretory neurons. Until the appearance of the thorough study of the preoptic nucleus of the goldfish C. auratus by Palay (16), no information was available on the fine structure of hypothalamic neurosecretory perikarya. Our observations on the preoptic nucleus of Rana are in accord with those of Palay in regard to the association of the typical elementary neurosecretory granules (in the 1000-1500-~ size range) with Golgi membranes. Of interest is the fact that the Golgi complex is considerably less prominent in verte-
320
HOWARD A. BERN ET AL.
brate as compared with invertebrate neurosecretory perikarya. Both the Dahlgren cells and the preoptic neurons of vertebrates show the same association between membranes and granules as do the invertebrate neurons, but the m e m b r a n e system is generally more diffuse in the former. We are indebted to Dr. Dorothy Pitelka for suggestions and for discussion of this material.
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