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AMER. ZOOI.., 14:551-573 (1974). Ultrastructural Studies of the Development of Nerves in Hydra LOWELL E. DAVIS Department of Biology, Syracuse University, Syracuse, New York 13210 SYNOPSIS. Three types of mature epidermal neurons and several of their differentiating stages aie presented in this ultrastructural study. Each of the three types, neurosensory, p.eurosecretory, and ganglionic cells, is derived from interstitial cells, (i) Mature neurosensory cells contain elongated nuclei, a well-developed cilium in each cell, and membrane-bounded neurosecretory droplets (700-1300A in diameter). There may be two or more neurites in which are numerous microtubules, glycogen particles, ribosoincs and many neurosecretory droplets, (ii) Mature neurosecretory cells closely resemble neurosensory cells, except that no cilium is present. The perikarya contain small, dens; nuclei, neurosecretory droplets (850-1300A in diameter), mitochondria, glycogen particles, and microtubules. Active Golgi complexes are present in both cell types. The nemites are similar to those described for neurosensory cells, (iii) Mature ganglionic celh are bipolar or multipolar. The small, dense nuclei are surrounded by a small amount of cytoplasm. The neurites contain mostly microtubules; a few mitochondria, ribosomes, and glycogen particles are also present, but there are no secretory droplets. To date, only neuios?nsory and neurosecretory cells have been observed in the gastiodermis. They are structurally indistinguishable from their epideimal counterparts. A significant finding is that three types of synapsjs—neuroinusciilar, neuronemalocyte, and inicrneuronal—are identified in both the epidermal and gastrodermal neurons. INTRODUCTION The nervous system of Hydra is composed of a gastrodermal and an epidermal plexus (Hyman, 1940). Little is known of the gastrodermal plexus except that a few investigators have referred to its existence (Hyman, 1940; Burnett and Diehl, 1964; Lent/., 1966), and recently, two types of neurons have been described and suggestions made concerning their origin (Davis, 1972). The epidermal nerve plexus is composed of three types of neurons, ganglionic, neurosensory, and neurosecretory cells (Burnett and Diehl, 1964; Lent/, and Barrnett, 1965; Davis et al., 1968). Each of these cells contains two or more neurites (bi-polar, tri-polar, or multi-polar) and as such they form a "continuous nerve net" extending throughout the epidermis. Like Some of the material presented in this paper was obtained as a result of a National Science Foundation Grant (CB-27395). The author also acknowledges the technical assistance of Linda M. Bookman. all other epidermal cells, neurons are sloughed off at the extremities (mouth, tentacle tips, and basal disk) and therefore must be replaced constantly. Previous ultrastructural studies on the development of neurons indicate that two of the three types of neurons—ganglionic and neurosensory cells—originate from interstitial cells (Lent/., 1965; Davis, 1969, 1971). Some of the crucial morphological stages of differentiation were presented, and it was suggested that despite certain structural similarities during early development, these two types of neurons originate directly and independently from interstitial cells. Furthermore, it was suggested that since neurons have never been observed in mitosis, the sole source of neurons is the interstitial cell. It has been assumed that the third type of neuron—the neurosecretory cell—has a similar independent origin from interstitial cells, but to date, there are no reports which demonstrate the development of this cell type. This paper will deal exclusively with the ultrastructural development and mor- 551 ,532 LOWELL E. DAVIS FIG. 1. Interstitial cell showing a centrally located nucleus and prominent nucleolus. The cytoplasm is filled with numerous ribosomes (ri) , few mitochondria and a few small vesicles. X 19,600. phology of fully differentiated neurons. It is emphasized that due to space limitations, only selected developmental stages and profiles of mature cells will be presented. propriate here to enumerate some general considerations. Although neurons are found throughout the epidermis, they are concentrated in such regions as the bases of tentacles, the hypostome, the peduncle, and the basal disk. Regardless of regional distribution they are located immediately adjacent to the longitudinal myonemes at GENERAL CONSIDERATIONS Before discussing the development of the individual types of neurons, it seems ap- ULTRASTRUCTURE OF HYDRA NERVES the bases of epithelio-muscular cells. In such locations, neurons are surrounded completely or partially by epithelio-muscular cells or partially by cnidoblasts, other neurons, and interstitial cells. Gastrodermal neurons are located at the bases of digestive cells, immediately adjacent to the circular myonemes. They are. therefore, surrounded mostly by digestive cells, and in some cases, may be partly surrounded by basal reserve cells. Secondly, neurites are usually tortuous and branched, and extend into different planes and directions. Accordingly, it is exceedingly difficult to obtain serial sections of entire cells. Due to certain structural similarities which will be demonstrated later, it is imperative that for accurate identification of the individual neurons, serial sections containing the nerve cell bodies (perikarya) be examined carefully. Finally, it should be realized that these studies represent a logical construction of the differentitive processes, based on the observation and interpretation of static electron microscopic images, are are therefore susceptible to some criticisms. DIFFERENTIATION OF EPIDERMAL NEURONS Interstitial cells Since interstitial cells are the undifferentiated cells from which neurons originate, it is appropriate to discuss briefly their ultrastructure so that a more meaningful comparison may be made between them and the early stages of neuronal development. They are usually round or oval, measuring approximately 5 to 6 tx in diameter (Fig. ]). They are located singly or in groups among epithelio-muscular cells and cnidoblasts. The nucleus, containing a conspicuous nucleolus, is centrally located and occupies a considerable portion of the cell. The cytoplasm is unquestionably embryonic in character, in that it contains numerous ribosomes, few small mitochondria, an inconspicuous Golgi complex, and few small vesicles, and shows a virtual 553 absence of rough endoplasmic reticulum. These cells undergo frequent mitosis and differentiate subsequently into neurons and to other cell types (e.g., cnidoblasts, spermatozoa and ova). Neurosen.sory cells Early developmental stages of the neurosensory cell are shown in Figures 2—4. The earliest recognizable stage reveals an ovalshaped appearance of the original interstitial cell and the presence of two centrioles (arrows in Fig. 2a) at one of the elongated cytoplasmic regions. From this point, the ciliary apparatus begins to develop. One of the centrioles (Fig. 2b) assumes a characteristic alignment immediately beneath the plasma membrane. As differentiation proceeds, the emerging ciliary bud (Fig. 2fZ) shows a striking resemblance to young ciliary processes of vertebrate neurons (Thornhill, 1967). The ciliary bud is surrounded completely by the plasma membrane and contains axial microtubules, a few dense particles dispersed among the microtubules, and an amorphous material. It has also been shown that while the cilium proper is being elaborated, electron-dense granules, 700 A in diameter, arranged in a linear fashion, extend inwardly from the base of the centriole (Davis, 1969). It is believed that these granules may be the precursor materials for initial rootlet formation shown in Figure 2c. The role of the Golgi complex and segments of rough endoplasmic reticulum which are located characteristically at the base of the developing cilium is unknown. As the ciliary shaft elongates into the extracellular space, internal microtubules extend its entire length (Fig. 3). In transverse sections they reveal the typical 9 + 2 arrangement of cilia, but the number and location of the tubules vary from one level to another (see Roth and Shigenaka, 1964; Satir, 1965; Boquist, 1968; Davis, 1969). Definite rootlet fibers appear at the base of the cilium, but they are not striated at this stage. The electron-dense granules LOWELL E. DAVIS FIG. 2. Development of the cilium in neurosensory cells, a, Two centrioles (arrows) oriented at right angles to each other near the periphery of the differentiating neurosensory cell. X24.400. b, Centriole (ce) aligned beneath the cell membrane, dense fibro-gianular materials (650-700 A in diameter) , and cross-section of niicrotubules (arrows) . X25.300. c, Fibers (arrows), believed to be precur- sors of rootlets, extend inward from the base of the centriole (ce). x 18,600. d, Ciliary bud protruding from the cell contains axial microtubules within an amorphous material. The developing cilium is surrounded completely by the plasma membrane. Definite rootlet fibers (ro) are observed for the first time, x25,300. which extended linearly from the base of the cilium (considered to be precursor materials for rootlet formation) have dis- appeared completely. The question regarding the number of cilia in each neurosensory cell remains un- U LTRASTRUCTURE OF HYDRA NERVES FIG. 3. Portions of two neurosensory cells (ns) , one of which contains a ciliiini (ci). Microtubules appear throughout the entire length of the ciliuin. Rootlets (10) are seen at the base of the cilium but at this stage they are not striated. Numerous ribosomes, Golgi complex (g) and short segments of rough endoplasmic reticulura (er) are also observed. X 19,900. FIG. 4. Golgi complexes in neurosensory cells, a, One Colgi complex (g) is located characteristically near the base of the developing ciliuin (ci) ; endoplasmic reticulum (er). b-d, Colgi complexes (g) in mature cells. Note the membrane-bounded secretory droplets (800-1000 A in diameter), lough ER (er) and larger irregularly shaped droplets (d). a, X25.800; b. X27.600; c, X41.300; d, X41.300. 556 LOWELL E. DAVIS resolved. Several investigators, notably McConnell (1932), Burnett and Diehl (1964), and Bullock and Horridge (1965), have indicated the presence of one to several "hairs" (cilia). Electron microscopical studies by Lentz and Barrnett (1965) and Davis et al. (1968) suggested only a single cilium in each neurosensory cell. More recent ultrastructural studies (Davis, 1969) and our investigations to date demonstrate that while more than one centriole may be directed toward ciliary development, there remains no definite ultrastructural evidence for bi- or multi-ciliated neurosensory cells. Prior to the complete maturation of the cilium, several cytoplasmic changes become evident. In addition to the elongation of the cytoplasm in various regions during the formation of neurites, certain organelles, particularly the Golgi complexes, show remarkable development (Fig. 4) . From the single inconspicuous Golgi complex of the original interstitial cell, there may be as many as four Golgi complexes in these cells. Materials of relatively high density appear within the increased Golgi lamellae. Peripheral vesicles eventually become distended and separate from the Golgi membranes. They are first observed in the perikarya and as the cell matures, they are transported throughout the cell body and neurites. These membrane-bounded droplets (700 to 1300 A in diameter), described as neurosecretory droplets, reveal a striking morphological similarity to comparable droplets in known neurosecretory cells of other organisms (Bern et al., 1962; Bonga, 1970; Andrews et al., 1971). Several other cytoplasmic changes also occur. The numerous ribosomes of the original interstitial cell persist throughout the formation of the cilium. Thereafter, their number diminishes and when the neurosensory cell is mature, most ribosomes have disappeared. Rough endoplasmic reticulum is not a prominent organelle in developing or differentiated cells. Only short segments are present and therefore it has been assumed that this organelle participates to a minor degree, if at all, in the synthesis of neurosecretory droplets. Mitochondria increase in number and usually become reduced in size. In most instances, there is a reduction in the number of cristae and an increase in the density of the matrix. Microtubules are first observed at the base of the developing cilium, and as the cell elongates during the formation of neurites, they extend for long distances, parallel to the long axis of the cell. The cell body of a mature neurosensory cell is shown in Figure 5. The nucleus, elongated or irregularly shaped, contains scattered dumps of chromatin material. The nucleolus which was once very conspicuous has fragmented to the point that it is not easily recognizable. The cytoplasm contains a characteristic cilium, typical neurosecretory droplets and larger ovalshaped droplets (up to 1.0 ^ in diameter), small dense mitochondria, few ribosomes and a Golgi complex. The cell body may also reveal synapses with other cells (e.g., cnidoblast shown in Figs. 5 and 6). These will be discussed in more detail later. Another profile of a mature neurosensory cell is shown in Figure 7. The cilium with its internal tubules is seen at greater advantage adjacent to the neurite of another nerve cell. Ncurosecrelory cells Morphological data concerning the differentiation of neurosecretory cells are extremely limited. All available evidence, however, indicates that these cells are also derived directly and independently from interstitial cells (Davis, 1971). An immature neurosecretory cell containing two neurites (top and bottom) is shown in Figure 8. As the cell elongates during the formation of neurites, the nucleus assumes an oval or elongated shape, and the perikaryon contains only a small amount of cytoplasm. Ribosomes, small mitochondria, and microtubules are observed in the cytoplasm of the cell body. At the base of the neurites, however, two or more Golgi complexes develop and are ULTRASTRUCTURE OF HYDRA NERVES FIG. f>. Mature neurosensory cell with cilium (ci) and associated structures, irregularly-shaped nucleus (nu) , Colgi complex (g) , dense membranehounded droplets (900-1100A in diameter), larger type droplets (d), and microtubules (mt) aligned 557 parallel to the long axis of the cell. Xote the crosssection of the flagellum and stereocilia of a nematocyst (ne) and points of contact (a and b) to the neurosensory cell. x21,400. 558 LOWELL E. DAVIS FIG. 6. Higher magnification of the contact (arrows) shown at (a) in Figure 5. Note the increased density of the plasma membranes and a droplet of moderate density in the neuroscnsory cell. Nematocyst (ne) in a cnidoblast (en). X 36,900. FIG. 7. Portion of a mature neurosensory cell showing elongated vesicles with dense chromatih material, fully-formed cilium (ci) , rootlet fibers (ro), small dense mitochondria (mi) , microtulmles (ml), and secretory droplets (900-1300 A in diameter) of various densities occupying only a portion of the space enclosed by the membranes. Neurites (neu) ; myonemes of epithelio-muscular cell (my) . X 19,000. aligned parallel to the long axis of the cell. Oriented in this fashion, some of the secretory droplets elaborated by the Golgi complex remain in the perikarya while others are transported into the neurites. The neurites at this stage also contain ele- ments of rough E.R., mitochondria, ribosomes, few microtubules, and large, irregularly-shaped droplets, the nature of which is still obscure. The perikaryon of a mature neurosecretory cell is shown in Figure 9. The nucleus ULTRASTRUCTURE OF HYDRA NERVES FIG. 8. Immature neurosecretory cell containing two neurites (only one shown). Golgi complexes (g) are located at the base o£ the neuiite in which there are mitochondria (mi) and short segments o£ 559 E.R. (ei) . Xote also the few membrane-bounded secretory droplets (1300 A in diameler) , larger droplets (d) , ribosoines, and inicrotubules (nil) . X 22,000. 500 LOWELL E. DAVIS FIG. 9. Mature neurosecretory cell. The nucleus contains scattered dumps of chromalin material. There are several dense membrane-bounded droplets (850-1300 A in diameter), small dense mito- chondria (mi) , microtubules (nit) , Golgi complex (S) • glycogcn particles (gl) , and larger dense droplets, up to 0.6 /i in length (d) . Myonemes (my) of epithelio-muscular cells (cp) . X26.200. reveals obvious changes from the immature nucleus in that there are scattered clumps of chromatin material and electron-lucent areas in the nucleoplasm. Typical membrane-bounded neurosecretory droplets are dispersed throughout the cell body. Their variation in size (850 to 1300 A in diameter) and density, as well as the presence of several empty vesicles, suggest that re- lease of the droplets also occurs in the perikaryon. T h e cytoplasm of this region also contains small dense mitochondria, large dense droplets, glycogen particles, Golgi complexes, numerous vesicles and vacuoles of various sizes, and microtubules which extend from the limits of the nuclear membrane into the neurites (Fig. 10). ULTRASTRUCTURE OF HYDRA NERVES FIG. 10. Portions of two mature neurosecretory cells (outlined) showing the nucleus (nu) and neurites (neu) . Xote the Colgi complex (g) at the base of the neurites, membrane-bounded secretory 561 droplets (800-1500 A in diameter) of various densities, empty vesicles (v), and microtubules (mt) . X 29,800. 562 LOWELL E. DAVIS FIG. 11. Neurites of neurosensory and/or neurosecretory cells are of various diameters. They contain primarily membrane-bounded droplets (8001300 A in diameter), microtubulcs (nil) , glycogen particles (gl), and mitochondria (mi) . Note (in 116) the specialized junctions (arrows) between a neurite and an epithelio-muscular cell (ep). i\fyonemes of epithelio-muscular cell (my) ; mesoglea (me) . a, X22/I0O; b, X32/I00; c, x39,800. 2 390 ULTRASTRUCTURE OF HYDRA NERVES S6B It has been indicated that neurites o£ neurosensory and neurosecretory cells are morphologically indistinguishable at the present lime. Furthermore, accurate identification of the individual neurites is possible only when the respective cell body is included in the section examined. Figure 11 shows three neurites which may belong to one or both mature cell types. Although FIG. 12. Immature ganglionic cell containing two cytoplasmic extensions (arrows). The mitochondria appear to accumulate at the bases of or within the extensions (neurites) and in two other areas which ma; also give rise to neurites. Except for the change in cell shape, the nucleus and cytoplasmic structures are similar to those of I-cells. Epitheliomuscular cell (ep). x 13,600. Neurites—neiiroscnsory fncurosecrctory cells LOWELL E. DAVIS they differ in terms of their ultimate length and diameter, they contain essentially the same components: membrane-bounded droplets (800 to 1300 A in diameter), microtubules, mitochondria, glycogen particles, and few ribosomes. Some of these neurites apparently end freely while others form specialized junctions (synapses) at their terminations (Fig. lie). • • * Ganglionic cells One of the earliest stages of ganglionic cell development is shown in Figure 12. Except for the presence of long cytoplasmic extensions, this cell type can be easily mistaken for an interstitial cell because of the obvious similarities. ]n addition, the problem is further compounded by the struc- < FIG. 13. Immature multi-polar ganglionic cell (outlined) with four neurites (arrows). The mi- clcolus is still recognizable. The cytoplasm contains numerous ribosomes. x30,500. ULTRASTRUCTURE OF HYDRA NERVES FIG. 14. A fully differentiated ganglionic cell (gc) . Note that the neurite contains mostly microtubules (mt) . Epithelio-muscular cell (ep) . x 13,900. tural similarity between these developing cells and both developing neurosensory cells and early cnidoblasts. In the case of the neurosensory cells, this early developmental stage involves the formation of the ciliary apparatus. On the other hand, cnidoblasts reveal developed Golgi com- plexes and the formation of several ves icles. This early stage of development, therefore, represents one of the most crucial stages to identify accurately. A slightly later developmental stage (outlined) is shown in Figure 13. The nucleus has now become elongated and LOWELL E. DAVIS FIG. 15. Immature gastrodermal neurosensory cell containing a cilium (ci) and a centriole (arrow). A cytoplasmic extension of the cell lies between the bases of digestive cells (dc) and is therefore immediately adjacent to the mesoglea (me) . Golgi complex (g); rough E.R. (er); epithelio-muscular cell (ep). x 22,200. FIG. 16. Ncurite of gastrodermal neurosensory or neurosecretory cell containing membrane-bounded droplets (750-1500A in diameter). Note the intramitochondrial crystal (arrow) . Myonemes (my) of digestive cell; mesoglea (me); epithelio-muscular cell (ep). x20,000. Ul-TRASTRUCTURE OF HYDRA NERVES 567 the nucleolus has begun to be dispersed throughout the nucleoplasm. The cyto- plasm, containing numerous ribosomes and a few mitochondria, extends in four dif- FIG. 17. Mature gastrodermal The nucleus, displaced to the tains dense clumps of scattered Active Golgi complexes (g), rough E.R. (er), microtubules (int) , small dense mitochondria (mi) , secretoiy droplets (1IOO-1600A in diameter), and larger dense droplets (arrows) are observed. X22,000. neurosecrelory cell. cell periphei), condiromatin material. short segments of 568 LOWELL E. DAVIS FIG. 18. Basal reserve cell in the gastroclermis. The cell resembles an epidermal interstitial cell: centrally located nucleus with a prominent niicleolus, numerous ribosomes (ri) , few elements of rough E.R. (er), and few mitochondria (mi). Long, nar- row cytoplasmic extensions (arrows) suggest that differentiation may have begun. Digestive cell (dc); mesoglea (me) ; epithelio-muscular cell (ep). X 22,200. I 569 ULTRASTRUCTURE OF HYDRA NERVES ferent directions (arrows) in the formation of a multipolar cell. From this stage in development up to the mature cell, one seldom sees more than two neurites in the same section. Further maturation of the cell involves the continued elongation of the neurites. Microtubules then begin to appear throughout the neurites, and by the time the cell is completely mature the neurites contain mostly microtubules, few mitochondria and ribosomes, and glycogen particles (Fig. 14). GASTRODERMAL NEURONS Exceedingly little is known concerning gastrodermal neurons except for the brief references as to their existence (Hyman, 1940; Burnett and Diehl, 1964; Lentz, 1966). Recently, however, we described two types of neurons in the gastrodermis: neurosensory and neurosecretory cells (Davis, 1972). The recognition was based on the structural criteria used for identification of epidermal neurons. An immature gastrodermal neurosensory cell containing neurites and a developing cilium is shown in Figure 15. One neurite has penetrated through the bases of digestive cells and lies immediately adjacent to the mesoglea. This represents an unusual observation since epidermal neurons have not been seen bordering the mesoglea. The neurites of mature neurosecretory and neurosensory cells (Fig. 16) contain typical membrane-bounded secretory droplets (750 to 1500 A in diameter), glycogen particles, and mitochondria. Neurosecretory cells are the second type of neuron found in the gastrodermis. A mature cell is shown in Figure 17. The nucleus, with dense chromatin material dispersed throughout the nucleoplasm, and all the cytoplasmic components (active Golgi complexes, secretory droplets [110 to 1600 A in diameter], mitochondria, microtubules, rough E.R.) are identical in structure to their epidermal counterparts. From the sparse available data, it appears that gastrodermal neurons originate from the basal reserve cells. These cells are located immediately adjacent to the circular myonemes at the bases of digestive cells or may lie deeper within the gastrodermis. Accordingly, they are surrounded completely by digestive cells or partly by digestive cells and neurons. Figure 18 shows a basal reserve cell which may have begun to differentiate into a nerve cell. The nucleus is centrally located and contains a prominent nucleolus. The cytoplasmic contents resemble those of interstitial cells in that there are numerous free ribosomes, few mitochondria, and short segments of rough E.R. It is noted that these cells sometimes contain large droplets ranging up to 0.7 /x in diameter. This structural similarity with interstitial cells, together with the fact that both cell types are capable of division and differentiation into other cell types (Rose and Burnett, 1968, 1970), allows one to consider the basal reserve cells as embryonic cells. Accordingly, these cells seem to be the most likely cell type from which gastrodermal neurons originate. SYNAPSES Most investigators concerned with the morphology of the nervous system of Hydra have been cautious in stating that synapses have not been observed in this organism. Our maceration studies of stained tissues show repeatedly that there are intimate associations between neurons, between neurons and epithelio-muscular cells, and between neurons and cnidoblasts. Ultrastructural studies later confirmed the definite existence of synapses. Westfall et al. (1971) provided evidence for three types of synapses, interneuronal, neuromuscular, and neuronematocyte, in the epidermal nerve net. We will now present briefly some evidence for synapses. Figure 19a shows two gastrodermal neurites (a and b) located adjacent to each other and between the circular myonemes of digestive cells. They contain typical membrane-bounded droplets (900 to 1800 A in diameter), glycogen 570 LOWELL E. DAVIS MG. 19. a, Two neurites (a and b) of gaslrodermal neurosensory and/or ncurosecretory cells located near the base of a digestive cell (dc) . The droplets are 990-1800 A in diameter. Note the point of contact between the two neurites (ins) and between neurite and myonemes (nms) of digestive cell (dc) . Mesoglea (me); epithelio-muscular cell (ep). x 16,000. b, Higher magnification of the neiiromiisrular synapse (nms) and interneuronal synapse (ins) shown in Figure 19a. Note ihe thickened membranes, vesicles (1100-1500 A in diameter) containing dense droplets (900-1100 A in diameter) and synaptic clefts (approximately 100 A wide). Myonemes (my) of digestive cell (dc). X 51,800. ULTRASTRUCTURE OF HYDRA NERVES FIG. 20. Synapses (arrows) in epithelial neurons. a, Synapse at perikaryon of neurosensory cell (ns) and epithelio-muscular cell (ep) . Nucleus (nu) . X 70,000. b, Synapse (arrows) between neurite (neu) and myoneme (my) of epithelio-rauscular cell, x 35,700. c, Synapse (arrows) between neurite 571 (neu) and non-myoneme portion of epitheliomuscular cell (ep). Microtubules (mt); glycogen (gl) . X 26,000. d. Grazing section of two neurites (a and b) with interneuronal synapse (ins) and one (a) with neuromuscular synapse (nms) . Microtubules (mt) ; glycogen (gl) . X 35,700. 572 LOWELL E. DAVIS particles, mitochondria, and microtubules. Higher magnification (Fig. 196) of the same neurites shows that one neurite (center) contains a neuromuscular and an interneuronal synapse. The vesicles at these junctions still contain dense contents. The interneuronal synapse also shows thin filaments which bridge the interneuronal gap. Figure 20 demonstrates the existence of synapses in the epidermal neurons. They may occur at the perikaryon of the cell (Fig. 20a), at neurite terminations (Figs. lib, 20b) or along the length of the neurites (Fig. 20c-d). In all instances, the membranes at the junctions are thickened and there are vesicles of various sizes and densities. This evidence is still preliminary, but we are presently focusing our attention on a more detailed study of synapse development, location, structure and function. CONCLUSION The introduction of electron microscopy in the study of neurons in Hydra has led to several significant landmarks: (i) the reconfirmation of the definite existence of neurons (despite recent statements to the contrary); (ii) the discovery of neurosecretion, indicating that Hydra are the most primitive organisms in which neurosecretion has been demonstrated; and (iii) the discovery of synapses. These gains, however, have created a spectrum of problems, all of which are fertile areas of investigation. We need, for example, a better understanding of the ultrastructure of neurons, including synapses. Other areas for investigation should and must include the chemical nature of the neurosecretory substance, its synthesis, transport, mechanisms of release and functions; the specific functions of individual types of neurons; the role of neurons in integrating the animal's behavior; and the structural and functional changes during such processes as growth, regeneration, budding, initiation of sexuality, and aging. These problems indicate the trend of future research on neurons in Hydra. REFERENCES Andrews, P. M., D. E. Copeland, and M. Fingerman. 1971. Ultrastructural study of the neurosecretory granules in the sinus gland of the blue crab, Callinectes sapidus. Z. Zellforsch. 113:461471. Bern, H. A., R. S. Nishioka, and E. R. Hagadorn. 1962. Neurosecretory granules and the organelles of neurosecretory cells. In H. Heller and R. B. Clark [ed.], Neurosecretion. 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