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Mesenchyme cells in Fasciola hepatica (platyhelminthes): Primitive glia?
TISSUE AND CELL, 1994 26 (1) 12f131 IQ 1994 Longman Group UK Ltd.
SUZANNE
C. SUKHDEO
and MICHAEL V. K. SUKHDEO
MESENCHYME CELLS IN FASCl0f.A HEPATlCA (PLATYHELMINTHES): PRIMITIVE GLIA? Keywords:
Brain, primitive
glia, mesenchyme,
flatworm,
parasite,
ganglion
ABSTRACT. Mesenchyme cells and their processes are found in the cerebral ganglia of the parasitic flatworm. Fasciola heparica. The mesenchyme cell processes are found in two specialized associations within the ganglion: (i) as lamellac-like multilayer sheaths encircling the cerebral ganglia and separating it from the surrounding parenchyma ceils, and (ii) invaginated into the surface of large diameter (‘giant’) nerve processes to form trophospongium-like relationships. Based on morphological criteria, these mesenchyme cells resemble general invertebrate glial cells suggesting that the mesenchyme cells of these flatworms may rcprcsent the earliest glial-like cell.
Introduction
of F. hepatica and the free-living flatworm Notoplana acticola has shown that nonneuronal tissues of the ganglia may share some characteristics of glial cells of higher invertebrates (Koopowitz and Chien, 1974; Sukhdeo et al., 1988). The identification of ghal cells in lower invertebrates has been problematic largely because the original criteria were developed for vertebrates, and later, for higher invertebrates (Roots, 1978). For example, some of these criteria include establishing the embryologic origins of the cells, identifying specific morphological features (such as the presence of filaments and the nature of the anatomical relationships between the glial and nerve cells) and determining the physiological functions or roles of the glial cells in the nervous system (Roots, 1978; Radojcic and Pentreath, 1979). These criteria are particularly difficult to fulfil in the case of lower invertebrates like the flatworms because these organisms are acoelomate and it is difficult to differentiate between neuronal, glial and general packing (parenchyma) cells (Radojcic and Pentreath, 1979). The resulting confusion is reflected in the wide variation of terminologies to describe putative glial cells in the lower invertebrates. For example, parenchyme. mesenchyme, neuroglia and support cells have all been used to describe the non-nervous cells that are found associated with the cerebral ganglia or nerve
Fusciolu hepatica is a parasitic flatworm that
belongs in the phylum Platyhelminthes. The flatworms are interesting from an evolutionary viewpoint because these organisms are the first to have bilaterally symmetrical bodies, and a true central nervous system with a distinct and well-defined brain. Therefore, these animals represent a crucial step in the evolution of nervous systems. The general organization of the cerebral ganglia (‘brain’) and the neurocytology of the neural cell bodies and their processes in both freeliving and parasitic flatworms do not differ greatly from that of higher invertebrates (Koopowitz and Chien, 1974; Koopowitz, 1982; Sukhdeo et al., 1988). One large difference between flatworms and higher invertebrates is that glial cells are thought to be absent from the central nervous system of flatworms but make their first appearance in the next higher phylum, Nematoda, the roundworms (Bullock and Horridge, 1965; Radojcic and Pentreath, 1979). However, ultrastructural studies of the cerebral ganglia
Department of Animal Sciences, Rutgers, The State Umversity of New Jersey, Cook College, New Brunswick, New Jersey 08903-0231, USA. Received 31 August 1993 Revised 27 September 1993 Accepted 5 October 1993 123
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tracts of flatworms (Bullock and Horridge, 1965). It is clear that the nature and/or presence of glial cells in the lower invertebrates require clarification. This study is a first step in this direction. The purpose of this study was to characterize the cytological and anatomical interrelationships of non-neuronal cells associated with the ganglia of the parasitic flatworm Fasciola hepatica. Materials and Methods Adult Fasciola hepatica were collected from the bile ducts of Wistar rats (Hilltop Labs., PA, USA) that had been infected 4-6 months previously with 15-20 metacercariae (the infective stage). The metacercariae were purchased from a commercial supplier (Baldwin Enterprises, Oregon, USA). The worms were kept in a buffered saline solution (pH 7.9) (Sukhdeo et al., 1986) for not more than 5 min prior to fixation. Adult worms measured 1.5-2.0 cm in length. The anterior region, approximately 2mm from the end, including the oral sucker and pharynx, was removed and placed in fixative for 90 min at room temperature. The fixative consisted of 1.5% or 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (pH 7.4) containing 0.1 M sucrose and 0.05% CaClz for about 90 min. The specimens were then postfixed in 1% 0~0~ in Millionig buffer (pH 7.4) for 1 hr and stained en bloc in 0.05% uranyl acetate in an acetate buffer (pH 5.4) for 1 hr. The specimens were dehydrated rapidly in a graded series of ethanols and embedded in Epon/Araldite. Gold serial sections were cut and counter-stained with uranyl acetate and
lead citrate. Sections were viewed with a Philips 201-C electron microscope. Results Two distinct cell types are found in the cerebral ganglion of F. hepatica. One is the neuron, and these elaborate either small (<12pm diameter), or giant (>12pm diameter) nerve processes. The second is a nonneuronal mesenchyme cell belonging to the broader parenchyma cell family. Mesenthyme cell bodies are morphologically distinct from neuronal cell bodies. They contain a densely stained nucleus with numerous aggregates of dense chromatin (Fig. 1). The cytoplasm is of a dense granular nature and contains large black inclusion bodies in addition to the golgi apparatus and mitochondria (Fig. 1). In contrast, the neuronal cell bodies have a lightly stained nucleus with a nucleolus (Fig. 2). The cytoplasm of the neuron cell body is also more lightly stained than the mesenchyme cell and contains the usual complement of organelles plus clear and dense vesicles (Fig. 2). Both mesenthyme and neuronal cell bodies are usually found around the periphery of the ganglion although mesenchyme cell bodies are often found among the nerve processes in the center of the ganglion (Fig. 3). The mesenchyme processes are also easily distinguished from neuronal processes by their staining characters, in particular the presence of the opaque inclusion bodies (Figs 3-5). A morphological character of the mesenchyme cells in the cerebral ganglia is that numerous long processes radiate from
Fig. 1. Mesenchyme cell body in Fasciola hepatica. The nucleus (N) is densely stained and contains numerous chromatin clumps (white arrows). The cytoplasm is also densely stained and contains mitochondria (m). golgi apparatus (small arrow) and opaque inclusion bodies (small arrowheads). Note, one of the opaque inclusion bodies is clearly seen in the invagination of a giant nerve cell process (large arrowhead). G, giant nerve process, x8300. Fig. 2. Neuronal cell body in Fascio~o hepatica. A large lightly stained nucleus (N) usually with only a single stained nucleolus. The stained cytoplasm is lighter when compared to the mesenchyme cell cytoplasm (compare with Fig. l), and contains numerous mitochondria and golgi apparatus but no black inclusion bodies. m, mitochondria. ~5400. Fig. 3. Transverse section through a cerebral ganglion of F. hepatica embedded in parenchyme cells (P). The central core of the ganglion, the neuropile (Np) is surrounded by several mesenchyme cell bodies (M). A few neuronal cell bodies (N) are also seen in this section. Separating the neuronal processes from the parenchyme cells are multi-layer stacked lamellaelike sheath or capsule (arrows). Mesenchyme processes run throughout the ganglion and can be easily distinguished by the opaque inclusion bodies (arrowheads). Mu, muscle fibers. x3000.
MESENCHYME
CELLS IN A FLATWORM
the cell body. These cell processes are found associated with the ganglion in two distinct locations, encircling the ganglion and invaginating into giant nerve cell processes. The mesenchyme processes that are found around the ganglion appear as many thinly stacked lamellae-like processes (Figs 3,4). These processes completely encircle the ganglion separating neuronal processes from the surrounding packing (parenchyma) cells. These mesenchyme cells also send their processes within the ganglion (Figs 3-5) to enclose clusters of neuronal processes (Fig. 5) although consistent organization into distinct regions cannot be discerned. The mesenchyme processes are also found associated with giant nerve cells in the neuropile. The giant nerve processes have highly convoluted cell membranes and the invaginations are filled by the mesenchyme cell processes (Fig. 6). Some of the invaginations clearly contain darkly stained cytoplasm of mesenchyme cells, containing the opaque inclusion bodies (Figs 1,6). The invaginations into the giant nerve cells are not only found in its processes but also into the cell body itself (Fig. 7). The giant neuron cells send off very large diameter processes (Fig. 8) that run through the neuropile and coalesce into the longitudinal nerve cords and commissures. The giant processes are, apart from size, similar to the smaller nerve
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processes in that they contain microtubules and vesicles (Fig. 9). In summary, the relationship between the giant neurons and the mesenchyme cells is close, intimate and often reciprocal (Fig. 10). Discussion Flatworms are acoelomate and lack a body cavity. A loose tissue network, the parenchyma which includes the mesenchyme cells, fills the spaces between internal organs and the body wall. The parenchyma is thought to act like a vascular system, collecting nutrients, metabolites and waste products (Gallagher and Threadgold, 1967). In addition, the mesenchyme cells of the parenchyma may function as energy storage sites; they contain large amounts of LY-and Pglycogen and are capable of both glycogenolysis and glycogenesis (Threadgold and Gallagher, 1966; Threadgold and Arme, 1974). In regions where the parenchyma interfaces with organs, the mesenchyme cells elaborate ‘pseudopodia-like’ processes that penetrate and interdigitate with the epithelial cells of these tissues. This is seen in interactions with the alimentary and excretory systems, and it is believed that these elaborations may facilitate the transport function of the mesenchyme cells (Gallagher and Threadgold, 1967).
Fig. 4. Mesenchyme (M) cell processes form the thin multi-layer stacked lamellae (large arrowheads) separating the packing parenchymes cells (P) from the neuronal processes of the cerebral ganglion (Cg). Note the stacked processes also contain numerous black inclusion bodies and these processes penetrate into the ganglion (small arrowheads). x2800. Fig. 5. A number of small nerve processes (*) bundled together by mesenchyme process in the cerebral ganglion. Mesenchyme processes (arrowheads) are clearly seen running through the neuropile area (Np). black circles, synapses and G, giant nerve process. x7100. Fig. 6. Cross-section through a giant nerve process showing the association with a mesenchyme process. The small arrowhead shows the tip of a mitochondria while the large arrow head points to two stacked mesenchyme processes in the invagination of the giant nerve process. arrows, microtubules and curved arrow, black inclusion bodies of mesenchyme origin. x 16.000. Fig. 7. Neuronal cell body (N) is completely surrounded processes (arrowheads). ~5700.
and invaginated by mesenchyme
Fig. 8. A giant neuron cell body (G) (the same one shown in Fig. 7) and part of its giant cell process protruding into the ganglion and bifurcating (arrowhead). Note the large number of opaque inclusion bodies in the surrounding cytoplasm. Np, neuropile. x3400. Fig. 9. A higher magnification of the giant nerve process in Figure 8 showing the abundant microtubules (arrows) and dense vesicles (arrowhead). x8700.
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Fig. 10. A mesenchyme cell body (m) enveloped by a giant neuronal process (G). The opaque inclusion bodies (arrowheads) of the mesenchyme processes are clearly seen as they, in turn, surround the giant neuronal process. x5400.
The results of the present study show that the mesenchyme cells of F. heparica have developed specialized elaborations around and inside the cerebral ganglion of this organism. There are two distinct mesenchymeneuron interactions that suggest glial-like functions. These are: (a) mesenchyme processes that surround the ganglia, and (b) mesenchyme processes associated with giant nerve processes. The cytology and morphology of the mesenchyme-neuron interactions are similar to glia-neuron interactions of higher invertebrates (Johnston and Roots, 1972; Radojcic and Pentreath, 1979). However, two major characteristics of glia are that they originate from embryonic ectoderm and that they have a close anatomical relationship with neurons (Radojcic and Pentreath, 1979). Mesenchyme tissue in this flatworm is of mesodermal origin and so fails the first requirement. However, information on glia is far from complete for any phylum and the embryological origin of glia is known in only two invertebrate phyla, the Annelida
and the Arthropoda, where the cells are derived from ectoderm (Roots, 1978). In F. hepatica, the mesenchyme cells fulfil the criteria for close anatomical association with nervous tissue. The ganglia of most invertebrates are surrounded by a capsule that consists of an inner layer of cells and an outer non-cellular layer (Radojcic and Pentreath, 1979). The evolutionary origin of this capsule is unclear but it is likely that it may have originated in the flatworms. Not all flatworms have a capsule or non-cellular sheath around their ganglia and the possession of this feature has been used to determine their phylogenetic positions within the phylum (Bullock and Horridge, 1965). In higher phyla, specialized glial cells, the perilemma cells, are responsible for secreting the layers of the sheath around the ganglia (Radojcic and Pentreath, 1979; Saint Marie et al., 1984). For example, the cortical glia of crustaceans is characterized by numerous stacked processes around the ganglia (Abbott, 1971) that are morphologically
MESENCHYME
CELLS IN A FLATWORM
similar to the loosely stacked mesenchyme processes that encircle the ganglia of F. hepafica. While this similarity does not impute similar function, it is noteworthy that the secretion of ganglionic sheaths and capsules in invertebrates is a glial cell function (Radjocic and Pentreath, 1979). One of the distinguishing features of all glial cells is the presence of extensive cell processes. In the neuropile, the compartmentalization of nerve fibers into tracts or glomeruli by glial processes is considered to be one of the essential functions of glial cells. As brains became increasingly larger and more complex, there was a need for specialized cells, i.e., glia, to organize and segregate (Bullock and Horridge, 1965). The removal of glial cells during development in Manduca sexta prevents the normal transformation of protoglomeruli into glomeruli, thus disrupting the normal synaptic function of the ganglion (Tolbert and Oland, 1990). Although mesenchyme processes enter into the neuropile and are found to enclose groups of nerve processes, there is no obvious segregation or compartmentalization of the ganglion of F. hepatica. However, this type of penetration of non-neuronal cell processes into the neuropile probably represents the earliest example of compartmentalization of the brain. The most convincing evidence for the glialike functions of the mesenchyme cells is seen in the interrelationships with giant nerve processes in the ganglia of F. hepatica. This relationship appears to be morphologically identical to trophospongium. Trophospongium describes the region of interactions between a neuron and its surrounding glial cell(s) where the glial cells invaginate into the neuron (Bullock and Horridge, 1965; Hoyle et al., 1986). It is a relatively widespread phenomenon in invertebrate nervous systems and is particularly well-developed in insects, molluscs and annelids where neuron cell bodies and axions may be ensheated by 2-20 layers of processes from surrounding neuroglia cells with the innermost layer penetrating into the neuronal cytoplasm (Bullock, 1961; Amoroso et al., 1964; Johnston and Roots, 1972; Pentreath et al., 1974; Nolte et al., 1976). The function of the trophospongium is thought to be trophic in nature but many additional roles have been suggested (see Nolte et al., 1976; Saint Marie
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and Carlson, 1983; Hoyle, 1986; Hoyle et al., 1986; Pentreath, 1989). In F. hepatica, the mesenchyme cell processes that invaginate into the giant neuron cell bodies may function like the trophospongium of higher invertebrates but there is as yet no empirical evidence of functional physiology. Thus far, trophospongium-like features associated with the CNS have not been reported from studies of other members of the flatworm phyla (Dixon and Merca, 1965; Koopowitz and Chien, 1974; Webb and Davey , 1975). Glial cells, in particular, astrocytes, possess the same kinds of voltage-dependent ion channels as neurons (Barres et al., 1990), and many express one or more types of ligandgated channels in response to neurotransmitters, e.g. glutamate (Usowicz et al., 1989) and GABA (Bormann and Kettenmann 1988). In addition, glial cells, such as the Schwann cells of squid giant axon, can synthesize and release neurotransmitters, such as acetylcholine (Heumann et al., 1981). In an earlier study on the histochemical localization of acetylcholinesterase in F. hepatica, a darkly stained ganglionic cell was identified as having acetylcholinesterase reaction product in its golgi apparatus (Sukhdeo et al., 1988). This acetylcholinesterase-containing cell is identical to the mesenchyme cell described in this study both in morphology and location. One of the phylogenetically oldest neurotransmitters is acetylcholine (Michelson and Ziemal, 1973) and acetylcholine has been shown to have a demonstrable effect on motor activity in this parasite (Sukhdeo et al., 1986). Thus, this data may constitute the first evidence of physiological function for the mesenchyme cell of a flatworm. A clarification of the identify of true glia in invertebrate nervous system is not within the scope of this paper. It is generally considered that the glia of invertebrates present a tremendous variety of morphological classes and types so that it is impossible to classify them based on generalizations (Johnston and Roots, 1972). Glia in the lower invertebrates may have evolved as specific functional adaptations independent of embryological origin. We suggest that in the primitive acoelomate, the mesenchyme cells may have been ‘pre-adapted’ to some specific glial functions, e.g., nutrient transfer of
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acetylcholinesterase activity, and thus may represent the first glia cells. Acknowledgements
This study was supported
by grants from
Whitehall Foundation and the New Jersey Agricultural Experiment Station (D-061883-93). The authors would also like to express their thanks to John Grazul and the- EM facilities at the Bureau of Biological Research, Rutgers University.
References Abbot, N. A. 1971. The organization of the cerebral ganglion in the shore crab, Car&us maenas. Zeit. Ze[l. Anat., 120, 386-400. Amoroso, E. C., Baxter, M. 1. and Chiquoine, A. D. 1964. The fine structure of neurons and other elements in the nervous system of the giant African land snail Archachatina marginata. Proc. Roy. Sot. Land. Series B, 160, 167180. Barres, B. A. Chun, L. L. Y. and Corey, D. P. 1990. Ion channels in vertebrate glia. Annu. Reu. Neurosci., 12,441414. Bormann, J. and Kettenmann, H. 1988. Patch-clamp study of GABA receptor Cl channels in cultured astrocytes. Proc. Natl. Acad. Sci. USA, 85, 93369340.
Bullock, R. H. 1961. On the anatomy of the giant neurons of the visceral ganglion of Aplysia. In Nervous Inhibition. Proceedings of the Second Friday Harbor Symposium (ed. E. FIorey), pp. 233-240. Pergamon Press, New York. Bullock, T. H. and Horridge, G. A. 1965. Structure and Function in the Nervous Systems of fnuertebrates. Vol. 1, pp. 536595. Freeman & Co., San Francisco. Dixon, K. E. and Mercer, E. H. 1965. The tine structure of the nervous system of the cetcaria of the liver fluke, Fasciola hepatica L. J. Parasirol., 51, 967-976. Gallagher, S. S. E. and Threadgold, L. T. 1967. Electron microscope studies of Fasciola hepatica. II. The interrelationship of the parenchyma with other organ systems. Parasitology, 57, 627-632. Heumann, R., Villegas, J. and Herzfeld, D. W. 1981. Acetylcholine synthesis in the Schwann cell and axon in the giant nerve fiber of the squid. J. Neurochem., 36,765-768. Hoyle, G. 1986. Glial cells of an insect ganglion. J. Comp. Neural., 246,85-103. Hoyle, G., Williams, M. and Phillips, C. 1986. Functional morphology of insect neuronal cell-surface/glial contacts: The trophospongium. J. Comp. Neurol., 246, 113-128. Johnston, P. V. and Roots, B. 1. 1972. Neroe Membranes. Pergamon Press, Toronto. Koopowitz, H. 1982. Free-living platyhelminthes. In Electrical Conduction and Behauiour in’Simp[e’ Inuertebrares (ed. G. A. B. Shelton), pp. 35%392. Clarendon Press, Oxford. Koopowitz, H. and Chien, P. 1974. Ultrastructure of the nerve plexus in flatworms. I. Peripheral organization. Cell Tiss. Res., 155, 337-351. Michelson, M. J. and Zeimal, E. V. 1973. The function of the cholinergic synapse. In Acetylcholine. An approach to rhe Molecular Mechanism of Action. Pergamon Press, New York. Nolte, A., Reinecke, M., Kuhlmann, D., Speckmann, E.-J. and Schultze, H. 1976. Glial cells of the snail brain some remarks on the morphology and function. In Neurobiology of Inuertebrates. Gastropod Brain (ed. J. Salanki), pp. 123-138. Akademiai Kiado, Budapest. Pentreath, V. W. 1989. Invertebrate glial cells. Comp. Bioc. Phys., 93A, 77-83. Pentreath, V. W., Berry, M. S. and Cottrell, G. A. 1974. Anatomy of the giant dopamine-containing neurone in the left pedal ganglion of Planorbis corneus. Cell Tiss. Res., 151, 369-384. Radojcic, T. and Pentreath, V. W. 1979. Invertebrate glia. Prog. Neurobiol., 12, 115-179. Roots, B. I. 1978. A phylogenetic approach to the anatomy of glia. In Dynamic Properties of Glio Cells (eds. E. Schoffeniels, G. Franck, L. Hertz and D. B. Tower), pp. 45-54. Pergamon Press, Toronto. Saint Marie, R. L. and Carlson, S. D. 1983. The fine structure of neuroglia the lamina ganglionaris of the housefly, Musco domestica L. _I. Neurocytol., 12, 213-241. Saint Marie, R. L., Carlson, S. D. and Chi, C. 1984. The glial cells of insects. In Insect Ultrasrructure (eds. R. C. King and H. Akai), Vol. 2 pp. 435-475. Plenum Press, New York. Sukhdeo, S. C., Sangster, N. C. and Mettrick, D. F. 1986. Effects of cholinergic drugs on longitudinal muscle contractions of Fasciola hepatica. J. Parasitol., 72, 858-864. Sukhdeo, S. C., Sukhdeo M. V. K. 1988. Histochemical localization of acetylcholinesterase in the cerebral ganglia of Fnsciola hepntica, a parasitic flatworm. J parasitol., 74, 1023-1032. Sukhdeo, S. C., Sukhdeo, M. V. K. and Mettrick, D. F. 1988. Neurocytology of the cerebral; ganglion of Fasciola heparica (Platyhelminthes). .I. Camp. Neurol., 278, 337-343. Threadgold, L. T. and Arme, C. 1974. Electron microscope studies of Fasciola hepatica. Expr. Parasitol., 35, 38% 405.
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Thrcadgold. L. T. and Gallagher, S. S. E. 1966. Electron microscope studies of Fascmla hepurim. 1. The ultrastructure and relationship of the parenchymal cells. Parasitology, 56, 29%304 Tolbert, L. P. and Oland, L. A. 1990. Glial cells form boundaries for dewloping insect olfactory glomcruli. &r. Neural., 109, 19-28. Usowicz. M. M., Gallo, V. and Cull-Candy, S. G. 1989. Multiple conductance channels in type-2 cerebellar astrocytea activated by excitatory amino acids. Nature. 339, 380-383. Webb. R. A. and Davey. K. G. 1975. The gross anatomy and histology of the nervous system of the metaccstode ot Hymenolepis mirostoma. Can. J. Zooi., 53, 661-677.
SUZANNE
C. SUKHDEO
and MICHAEL V. K. SUKHDEO
MESENCHYME CELLS IN FASCl0f.A HEPATlCA (PLATYHELMINTHES): PRIMITIVE GLIA? Keywords:
Brain, primitive
glia, mesenchyme,
flatworm,
parasite,
ganglion
ABSTRACT. Mesenchyme cells and their processes are found in the cerebral ganglia of the parasitic flatworm. Fasciola heparica. The mesenchyme cell processes are found in two specialized associations within the ganglion: (i) as lamellac-like multilayer sheaths encircling the cerebral ganglia and separating it from the surrounding parenchyma ceils, and (ii) invaginated into the surface of large diameter (‘giant’) nerve processes to form trophospongium-like relationships. Based on morphological criteria, these mesenchyme cells resemble general invertebrate glial cells suggesting that the mesenchyme cells of these flatworms may rcprcsent the earliest glial-like cell.
Introduction
of F. hepatica and the free-living flatworm Notoplana acticola has shown that nonneuronal tissues of the ganglia may share some characteristics of glial cells of higher invertebrates (Koopowitz and Chien, 1974; Sukhdeo et al., 1988). The identification of ghal cells in lower invertebrates has been problematic largely because the original criteria were developed for vertebrates, and later, for higher invertebrates (Roots, 1978). For example, some of these criteria include establishing the embryologic origins of the cells, identifying specific morphological features (such as the presence of filaments and the nature of the anatomical relationships between the glial and nerve cells) and determining the physiological functions or roles of the glial cells in the nervous system (Roots, 1978; Radojcic and Pentreath, 1979). These criteria are particularly difficult to fulfil in the case of lower invertebrates like the flatworms because these organisms are acoelomate and it is difficult to differentiate between neuronal, glial and general packing (parenchyma) cells (Radojcic and Pentreath, 1979). The resulting confusion is reflected in the wide variation of terminologies to describe putative glial cells in the lower invertebrates. For example, parenchyme. mesenchyme, neuroglia and support cells have all been used to describe the non-nervous cells that are found associated with the cerebral ganglia or nerve
Fusciolu hepatica is a parasitic flatworm that
belongs in the phylum Platyhelminthes. The flatworms are interesting from an evolutionary viewpoint because these organisms are the first to have bilaterally symmetrical bodies, and a true central nervous system with a distinct and well-defined brain. Therefore, these animals represent a crucial step in the evolution of nervous systems. The general organization of the cerebral ganglia (‘brain’) and the neurocytology of the neural cell bodies and their processes in both freeliving and parasitic flatworms do not differ greatly from that of higher invertebrates (Koopowitz and Chien, 1974; Koopowitz, 1982; Sukhdeo et al., 1988). One large difference between flatworms and higher invertebrates is that glial cells are thought to be absent from the central nervous system of flatworms but make their first appearance in the next higher phylum, Nematoda, the roundworms (Bullock and Horridge, 1965; Radojcic and Pentreath, 1979). However, ultrastructural studies of the cerebral ganglia
Department of Animal Sciences, Rutgers, The State Umversity of New Jersey, Cook College, New Brunswick, New Jersey 08903-0231, USA. Received 31 August 1993 Revised 27 September 1993 Accepted 5 October 1993 123
SUKHDEO ET AL.
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tracts of flatworms (Bullock and Horridge, 1965). It is clear that the nature and/or presence of glial cells in the lower invertebrates require clarification. This study is a first step in this direction. The purpose of this study was to characterize the cytological and anatomical interrelationships of non-neuronal cells associated with the ganglia of the parasitic flatworm Fasciola hepatica. Materials and Methods Adult Fasciola hepatica were collected from the bile ducts of Wistar rats (Hilltop Labs., PA, USA) that had been infected 4-6 months previously with 15-20 metacercariae (the infective stage). The metacercariae were purchased from a commercial supplier (Baldwin Enterprises, Oregon, USA). The worms were kept in a buffered saline solution (pH 7.9) (Sukhdeo et al., 1986) for not more than 5 min prior to fixation. Adult worms measured 1.5-2.0 cm in length. The anterior region, approximately 2mm from the end, including the oral sucker and pharynx, was removed and placed in fixative for 90 min at room temperature. The fixative consisted of 1.5% or 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (pH 7.4) containing 0.1 M sucrose and 0.05% CaClz for about 90 min. The specimens were then postfixed in 1% 0~0~ in Millionig buffer (pH 7.4) for 1 hr and stained en bloc in 0.05% uranyl acetate in an acetate buffer (pH 5.4) for 1 hr. The specimens were dehydrated rapidly in a graded series of ethanols and embedded in Epon/Araldite. Gold serial sections were cut and counter-stained with uranyl acetate and
lead citrate. Sections were viewed with a Philips 201-C electron microscope. Results Two distinct cell types are found in the cerebral ganglion of F. hepatica. One is the neuron, and these elaborate either small (<12pm diameter), or giant (>12pm diameter) nerve processes. The second is a nonneuronal mesenchyme cell belonging to the broader parenchyma cell family. Mesenthyme cell bodies are morphologically distinct from neuronal cell bodies. They contain a densely stained nucleus with numerous aggregates of dense chromatin (Fig. 1). The cytoplasm is of a dense granular nature and contains large black inclusion bodies in addition to the golgi apparatus and mitochondria (Fig. 1). In contrast, the neuronal cell bodies have a lightly stained nucleus with a nucleolus (Fig. 2). The cytoplasm of the neuron cell body is also more lightly stained than the mesenchyme cell and contains the usual complement of organelles plus clear and dense vesicles (Fig. 2). Both mesenthyme and neuronal cell bodies are usually found around the periphery of the ganglion although mesenchyme cell bodies are often found among the nerve processes in the center of the ganglion (Fig. 3). The mesenchyme processes are also easily distinguished from neuronal processes by their staining characters, in particular the presence of the opaque inclusion bodies (Figs 3-5). A morphological character of the mesenchyme cells in the cerebral ganglia is that numerous long processes radiate from
Fig. 1. Mesenchyme cell body in Fasciola hepatica. The nucleus (N) is densely stained and contains numerous chromatin clumps (white arrows). The cytoplasm is also densely stained and contains mitochondria (m). golgi apparatus (small arrow) and opaque inclusion bodies (small arrowheads). Note, one of the opaque inclusion bodies is clearly seen in the invagination of a giant nerve cell process (large arrowhead). G, giant nerve process, x8300. Fig. 2. Neuronal cell body in Fascio~o hepatica. A large lightly stained nucleus (N) usually with only a single stained nucleolus. The stained cytoplasm is lighter when compared to the mesenchyme cell cytoplasm (compare with Fig. l), and contains numerous mitochondria and golgi apparatus but no black inclusion bodies. m, mitochondria. ~5400. Fig. 3. Transverse section through a cerebral ganglion of F. hepatica embedded in parenchyme cells (P). The central core of the ganglion, the neuropile (Np) is surrounded by several mesenchyme cell bodies (M). A few neuronal cell bodies (N) are also seen in this section. Separating the neuronal processes from the parenchyme cells are multi-layer stacked lamellaelike sheath or capsule (arrows). Mesenchyme processes run throughout the ganglion and can be easily distinguished by the opaque inclusion bodies (arrowheads). Mu, muscle fibers. x3000.
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the cell body. These cell processes are found associated with the ganglion in two distinct locations, encircling the ganglion and invaginating into giant nerve cell processes. The mesenchyme processes that are found around the ganglion appear as many thinly stacked lamellae-like processes (Figs 3,4). These processes completely encircle the ganglion separating neuronal processes from the surrounding packing (parenchyma) cells. These mesenchyme cells also send their processes within the ganglion (Figs 3-5) to enclose clusters of neuronal processes (Fig. 5) although consistent organization into distinct regions cannot be discerned. The mesenchyme processes are also found associated with giant nerve cells in the neuropile. The giant nerve processes have highly convoluted cell membranes and the invaginations are filled by the mesenchyme cell processes (Fig. 6). Some of the invaginations clearly contain darkly stained cytoplasm of mesenchyme cells, containing the opaque inclusion bodies (Figs 1,6). The invaginations into the giant nerve cells are not only found in its processes but also into the cell body itself (Fig. 7). The giant neuron cells send off very large diameter processes (Fig. 8) that run through the neuropile and coalesce into the longitudinal nerve cords and commissures. The giant processes are, apart from size, similar to the smaller nerve
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processes in that they contain microtubules and vesicles (Fig. 9). In summary, the relationship between the giant neurons and the mesenchyme cells is close, intimate and often reciprocal (Fig. 10). Discussion Flatworms are acoelomate and lack a body cavity. A loose tissue network, the parenchyma which includes the mesenchyme cells, fills the spaces between internal organs and the body wall. The parenchyma is thought to act like a vascular system, collecting nutrients, metabolites and waste products (Gallagher and Threadgold, 1967). In addition, the mesenchyme cells of the parenchyma may function as energy storage sites; they contain large amounts of LY-and Pglycogen and are capable of both glycogenolysis and glycogenesis (Threadgold and Gallagher, 1966; Threadgold and Arme, 1974). In regions where the parenchyma interfaces with organs, the mesenchyme cells elaborate ‘pseudopodia-like’ processes that penetrate and interdigitate with the epithelial cells of these tissues. This is seen in interactions with the alimentary and excretory systems, and it is believed that these elaborations may facilitate the transport function of the mesenchyme cells (Gallagher and Threadgold, 1967).
Fig. 4. Mesenchyme (M) cell processes form the thin multi-layer stacked lamellae (large arrowheads) separating the packing parenchymes cells (P) from the neuronal processes of the cerebral ganglion (Cg). Note the stacked processes also contain numerous black inclusion bodies and these processes penetrate into the ganglion (small arrowheads). x2800. Fig. 5. A number of small nerve processes (*) bundled together by mesenchyme process in the cerebral ganglion. Mesenchyme processes (arrowheads) are clearly seen running through the neuropile area (Np). black circles, synapses and G, giant nerve process. x7100. Fig. 6. Cross-section through a giant nerve process showing the association with a mesenchyme process. The small arrowhead shows the tip of a mitochondria while the large arrow head points to two stacked mesenchyme processes in the invagination of the giant nerve process. arrows, microtubules and curved arrow, black inclusion bodies of mesenchyme origin. x 16.000. Fig. 7. Neuronal cell body (N) is completely surrounded processes (arrowheads). ~5700.
and invaginated by mesenchyme
Fig. 8. A giant neuron cell body (G) (the same one shown in Fig. 7) and part of its giant cell process protruding into the ganglion and bifurcating (arrowhead). Note the large number of opaque inclusion bodies in the surrounding cytoplasm. Np, neuropile. x3400. Fig. 9. A higher magnification of the giant nerve process in Figure 8 showing the abundant microtubules (arrows) and dense vesicles (arrowhead). x8700.
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Fig. 10. A mesenchyme cell body (m) enveloped by a giant neuronal process (G). The opaque inclusion bodies (arrowheads) of the mesenchyme processes are clearly seen as they, in turn, surround the giant neuronal process. x5400.
The results of the present study show that the mesenchyme cells of F. heparica have developed specialized elaborations around and inside the cerebral ganglion of this organism. There are two distinct mesenchymeneuron interactions that suggest glial-like functions. These are: (a) mesenchyme processes that surround the ganglia, and (b) mesenchyme processes associated with giant nerve processes. The cytology and morphology of the mesenchyme-neuron interactions are similar to glia-neuron interactions of higher invertebrates (Johnston and Roots, 1972; Radojcic and Pentreath, 1979). However, two major characteristics of glia are that they originate from embryonic ectoderm and that they have a close anatomical relationship with neurons (Radojcic and Pentreath, 1979). Mesenchyme tissue in this flatworm is of mesodermal origin and so fails the first requirement. However, information on glia is far from complete for any phylum and the embryological origin of glia is known in only two invertebrate phyla, the Annelida
and the Arthropoda, where the cells are derived from ectoderm (Roots, 1978). In F. hepatica, the mesenchyme cells fulfil the criteria for close anatomical association with nervous tissue. The ganglia of most invertebrates are surrounded by a capsule that consists of an inner layer of cells and an outer non-cellular layer (Radojcic and Pentreath, 1979). The evolutionary origin of this capsule is unclear but it is likely that it may have originated in the flatworms. Not all flatworms have a capsule or non-cellular sheath around their ganglia and the possession of this feature has been used to determine their phylogenetic positions within the phylum (Bullock and Horridge, 1965). In higher phyla, specialized glial cells, the perilemma cells, are responsible for secreting the layers of the sheath around the ganglia (Radojcic and Pentreath, 1979; Saint Marie et al., 1984). For example, the cortical glia of crustaceans is characterized by numerous stacked processes around the ganglia (Abbott, 1971) that are morphologically
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similar to the loosely stacked mesenchyme processes that encircle the ganglia of F. hepafica. While this similarity does not impute similar function, it is noteworthy that the secretion of ganglionic sheaths and capsules in invertebrates is a glial cell function (Radjocic and Pentreath, 1979). One of the distinguishing features of all glial cells is the presence of extensive cell processes. In the neuropile, the compartmentalization of nerve fibers into tracts or glomeruli by glial processes is considered to be one of the essential functions of glial cells. As brains became increasingly larger and more complex, there was a need for specialized cells, i.e., glia, to organize and segregate (Bullock and Horridge, 1965). The removal of glial cells during development in Manduca sexta prevents the normal transformation of protoglomeruli into glomeruli, thus disrupting the normal synaptic function of the ganglion (Tolbert and Oland, 1990). Although mesenchyme processes enter into the neuropile and are found to enclose groups of nerve processes, there is no obvious segregation or compartmentalization of the ganglion of F. hepatica. However, this type of penetration of non-neuronal cell processes into the neuropile probably represents the earliest example of compartmentalization of the brain. The most convincing evidence for the glialike functions of the mesenchyme cells is seen in the interrelationships with giant nerve processes in the ganglia of F. hepatica. This relationship appears to be morphologically identical to trophospongium. Trophospongium describes the region of interactions between a neuron and its surrounding glial cell(s) where the glial cells invaginate into the neuron (Bullock and Horridge, 1965; Hoyle et al., 1986). It is a relatively widespread phenomenon in invertebrate nervous systems and is particularly well-developed in insects, molluscs and annelids where neuron cell bodies and axions may be ensheated by 2-20 layers of processes from surrounding neuroglia cells with the innermost layer penetrating into the neuronal cytoplasm (Bullock, 1961; Amoroso et al., 1964; Johnston and Roots, 1972; Pentreath et al., 1974; Nolte et al., 1976). The function of the trophospongium is thought to be trophic in nature but many additional roles have been suggested (see Nolte et al., 1976; Saint Marie
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and Carlson, 1983; Hoyle, 1986; Hoyle et al., 1986; Pentreath, 1989). In F. hepatica, the mesenchyme cell processes that invaginate into the giant neuron cell bodies may function like the trophospongium of higher invertebrates but there is as yet no empirical evidence of functional physiology. Thus far, trophospongium-like features associated with the CNS have not been reported from studies of other members of the flatworm phyla (Dixon and Merca, 1965; Koopowitz and Chien, 1974; Webb and Davey , 1975). Glial cells, in particular, astrocytes, possess the same kinds of voltage-dependent ion channels as neurons (Barres et al., 1990), and many express one or more types of ligandgated channels in response to neurotransmitters, e.g. glutamate (Usowicz et al., 1989) and GABA (Bormann and Kettenmann 1988). In addition, glial cells, such as the Schwann cells of squid giant axon, can synthesize and release neurotransmitters, such as acetylcholine (Heumann et al., 1981). In an earlier study on the histochemical localization of acetylcholinesterase in F. hepatica, a darkly stained ganglionic cell was identified as having acetylcholinesterase reaction product in its golgi apparatus (Sukhdeo et al., 1988). This acetylcholinesterase-containing cell is identical to the mesenchyme cell described in this study both in morphology and location. One of the phylogenetically oldest neurotransmitters is acetylcholine (Michelson and Ziemal, 1973) and acetylcholine has been shown to have a demonstrable effect on motor activity in this parasite (Sukhdeo et al., 1986). Thus, this data may constitute the first evidence of physiological function for the mesenchyme cell of a flatworm. A clarification of the identify of true glia in invertebrate nervous system is not within the scope of this paper. It is generally considered that the glia of invertebrates present a tremendous variety of morphological classes and types so that it is impossible to classify them based on generalizations (Johnston and Roots, 1972). Glia in the lower invertebrates may have evolved as specific functional adaptations independent of embryological origin. We suggest that in the primitive acoelomate, the mesenchyme cells may have been ‘pre-adapted’ to some specific glial functions, e.g., nutrient transfer of
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acetylcholinesterase activity, and thus may represent the first glia cells. Acknowledgements
This study was supported
by grants from
Whitehall Foundation and the New Jersey Agricultural Experiment Station (D-061883-93). The authors would also like to express their thanks to John Grazul and the- EM facilities at the Bureau of Biological Research, Rutgers University.
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