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Morphological organization of neuropile glial cells in the central nervous system of the medicinal leech (Hirudo medicinalis)
Tissue & Cell, 1998 30 (2) I77-186 © 1998 Harcourt Brace & Co. Ltd
Morphological organization of neuropile glial cells in the central nervous system of the medicinal leech (Hirudo medicinalis) B. Riehl, W.-R. Schlue
Abstract Neuropile glial (NPG) cells in the central nervous system of the medicinal leech, Hirudo medicinalis, were studied by histological, histochemical and immunocytochemical techniques. The NPG cells are often surrounded by electron-dense microglial cells. The central cytoplasm of NPG cells shows a significant zonation. The zone around the nucleus contains mitochondria, glycogen and vesicles. The cytoplasm also contains many ribosomes, a few dictyosomes and distinct inclusions up to 2 gm in diameter. A second zone around the perinuclear region is marked by the occurrence of bundles of intermediate filaments that correspond in thickness to glial filaments of vertebrates. We found a positive reaction with polyclonal antibodies against human glial fibrillary acidic protein (GFAP), and the areas of intense fluorescence correspond to the regions where intermediate filaments were found to be abundant. The peripheral zone contains numerous membrane stacks that could not be contrasted by lanthane nitrate or tannic acid. Therefore, the membrane stacks could be part of an extensive smooth endoplasmic reticulum, which is characteristic of cells with active lipid metabolism. Keywords: Leech,glial filaments, GFAP, ultrastructure,lipid content, neuropileglial cell
Introduction Glial cells in central nervous tissues of invertebrates are found at the border between hemolymph or blood and neuronal elements, where they surround neurons and their processes. It is difficult to compare morphological studies of glial cells in different invertebrates, because so many different glial cell types have been described on the basis of experiments using various markers (Radojcic and Pentreath, 1979). No morphological classification has been undertaken in invertebrates, and there is need for the definition of reliable characteristics (Roots, 1986). Institut for Neurobiologie, Heinrich-Heine-Universit~R Desseldorf, Universit&tsstr. 1, D-40225 DLisseldorf, Germany. Received 29 April 1997 Accepted 13 November 1997 Correspondence to: Dr Wolf-R0diger Schlue. Tel: (49) (211) 81 13414; Fax: (49) (211 ) 81 13279; E-mail: schlue @uni-duesseldorf.de.
The central nervous system of Hirudo medicinalis has been a standard preparation for electrophysiological experiments, because of its uniform and simple organization with good access to individual glial cells and neurons. The location, morphology and physiology of the neurons have been studied extensively (for review see Sawyer, 1986). In recent years there has been an increasing interest in the properties and functions of glial cells in the leech central nervous system (for review see Pentreath, 1989); however, the histological characterization of this type of cell is incomplete at present. Macroglial cells of the leech have been previously identified according to their location using electron microscopy (Gray and Guillery, 1963; Coggeshall and Fawcett, 1964) but these studies did not investigate the ultrastructure of neuropile glial (NPG) cells. These NPG cells have served as a model system with regard to the involvement of glial cells in processes of K +, H + and Ca 2+ regulation (Schlue and Wuttke, 1983; Schlue and Walz, 1984; Schlue et al., 1991; 177
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Hochstrate and Schlue, 1994). The NPG cells have also provided a suitable model system for investigating the ways in which certain neurotransmitters, i.e. 5-hydroxytryptamine, acetylcholine and glutamate, interact with receptors in the glial membranes so as to control the homeostasis of ions in the nervous system (Walz and Schlue, 1982; Ballanyi and Schlue, 1989; Dtrner et al., 1990, 1994; Hochstrate and Schlue, 1994). The cell bodies of NPG cells are located in the ventral mid-region of each neuropile and their processes surround neuropile axons and synaptic regions. As demonstrated previously, NPG cells can be identified by their carbonic anhydrase II activity (Riehl and Schlue, 1990, 1993). In the present study we investigate the ultrastructural properties of NPG cells using conventional electron microscopy, to elucidate their significant relationship to the physiological function of these cells. We found no evidence for gap junctions between these cells but, according to their morphological properties and cytoplasmic contents, NPG cells could be involved in a number of different physiological processes. We show that a significant zonation is present in the central cytoplasm of NPG cells, and that the peripheral zone contains numerous membrane stacks which could not be contrasted by lanthane nitrate or tannic acid. Combining the conventional and fluorescence techniques, we also demonstrate that bundles of intermediate filaments in NPG cells contain glial fibrillary acidic protein (GFAP), which is characteristic of vertebrate astrocytes (Traub, 1985).
Materials and methods Tissue preparation for ultrastructural examination Adult medicinal leeches were sectioned and tissues were fixed in a mixture of 2% formaldehyde and 0.2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 1 h at 4°C. The chosen reagents allowed good preservation of tissue for light- and electron-microscope examination. We noted that concentrations of glutaraldehyde exceeding 0.2% damaged the ultrastructure of NPG cells, although the surrounding tissue was well preserved. It appeared that other mixture compositions did not reach NPG cells rapidly enough to prevent destruction of tissue. After washing with 0.1 M sodium cacodylate buffer, the tissue was postfixed in a solution of 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at 4°C. In addition we used postfixation solutions of 1% lanthane nitrate (Shaklai and Tavassoli, 1977) or 2% tannic acid (Futaesaku et al., 1972) as extracellular markers. After additional washing in 0.1 M sodium cacodylate buffer tissues were dehydrated in an ascending series of acetone and embedded in TAAB epoxide (Araldite). Semithin sections, 0.3-0.5%tm thick, were cut on a Reichert Ultracut and stained with an aqueous solution of 1% methylene blue/l% disodium tetraborate, 1:1. Silvergrey ultrathin sections were cut with a diamond knife, stretched by xylene and collected on pioloform (Merck)
coated copper grids. Sections were contrasted for 30 min in a methanolic solution of uranyl acetate, washed with distilled water and dried. Next we incubated preparations for 1 min in lead-II-citrate-trihydrate (Venable and Coggeshall, 1965). Photographs were made with a Zeiss EM 9S electron microscope and an Olympus BH-2 light microscope.
Histochemical detection of lipid content Frozen tissue sections were briefly dehydrated and covered by a 50% ethanol solution for 30 s. To stain for neutral lipids and lipoids, sections were dropped into a Sudan III solution according to Daddi (cf. Adam and Czihak, 1964) for 30-60 s at room temperature. After another 30 s in 50% ethanol and 2 min rinsing in distilled water, sections were mounted with glycerol-gelatine. For distinction between acidic and neutral lipids, sections were incubated in 1% aqueous Nile Blue sulfate at 50°C for 5 min, rinsed in distilled water at 60°C and stained again in 0.2% Nile Blue sulfate solution. After rinsing in distilled water, sections were prepared as described above. Immunocytochemical detection of GFAP Tissue was examined by the indirect immunofluorescence technique with FITC-coupled antiserum according to Sternberger (1979). Non-specific labeling was blocked by a 30-min treatment of sections with normal goat serum (1:50 in PBS at room temperature). This step was followed by incubation with anti-human GFAP serum from rabbits (Dakopatts) in solutions between 1:50 and 1:500 in PBS for 1 h at room temperature or overnight at 4°C. After washing three times in PBS buffer for 10 min at 4°C, the slides were incubated for 1 h at room temperature with goat anti-rabbit Ig coupled with FITC (Sigma) diluted 1:50 in PBS. After additional washing in PBS the sections were viewed and photographed in an Olympus BH2 fluorescence microscope using excitation filters EY-455 and BP-490, dichroic mirror 500 and barrier filter 0-515.
Results Morphology and lipid content of NPG cells A single leech ganglion is delimited by an outer capsule (Fig. 1). There is also an inner ganglion capsule enclosing axons and cell bodies, occasional neuron somata in the periphery of the neuropile, many microglial cells scattered through the neuropile, and two especially large glial cells lying next to the neuropile in the anterior and posterior regions of each ganglion, the NPG cells. Analysis of serial sections showed that the central cytoplasm of the NPG cells is located at the central midline in each ganglion. The somata of the cells send out projections that enter the neuropile and branch within it; here they could interact with the synapses. This morphological characteristic in itself makes the NPG cells an interesting candidate for studies of neuron-glia interactions, for in the leech (as in other invertebrates) the fundamental operations of the
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Fig. 1 Cross-section through a ganglion of Hirudo medicinalis. There is an outer and inner capsule of connective tissue (CT) dividing the region of packet glial cells (PG) and neurons (N) from the inner neuropile (NP) and from the neuropite glial cell (NPG). The neuropile contains axons (A) and four synaptic regions (s). Inside the cytoplasm of the neuropile glial cell are numerous lipid drops (arrows), which are also in the processes (arrowheads). Semithin section (Araldite, Azur II/Borax). Light microscopy (LM).
central nervous system, that is, synaptic transmission and integration, occur in the neuropile. There are four main processes of these cells, in addition to many small processes extending from the cell body of the NPG cell into the neuropile (Fig. 2). Two of the large processes run laterally parallel to the inner connective tissue and completely enclose the mass of axons in the neuropile. The two others lie medially, dorsal to the central NPG cytoplasm, and are close to the 'giant afferent neurons' in the central neuropile (Fernandez, 1978). We did not find any direct contacts between NPG and other macroglial cells. At the regions where the neuronal processes cross the inner capsule, they are surrounded outside the neuropile by the packet glial cells and inside by the NPG cells. The two glial cell types are separated from one another by connective tissue or by microglial cells (Fig. 5). The NPG cells are often surrounded by electron-dense microglial cells and their processes (Figs 2, 5). Like all macroglial cells of the leech, NPG cells have a nucleus
with dense, filamentous material at its outer envelope (Fig. 5). The nucleoplasm is light, with a similar appearance to the neuronal nuclei. The cytoplasm of NPG cells is more electron-dense than that of other leech macroglial cells, and shows a marked division into zones. In the zone around the nucleus mitochondria, glycogen and vesicles of different content and size are regularly distributed. The cytoplasm also contains many ribosomes and a few dictyosomes (Fig. 4). Even in light-microscope sections distinct inclusions with a diameter up to 2 gm can be seen (Fig. 1). In electron micrographs these inclusions did not appear to be limited by a membrane and their abundance depended on the nutritional status of the animals. Staining with Sudan III (Fig. 10) and Nile Blue sulfate confirmed that the inclusions are lipids within the NPG cytoplasm. We found these lipids to be of acidic and neutral types. A second zone around the perinuclear zone described above was marked by the occurrence of many small bundles of intermediate filaments, in an annular arrangement (Fig. 3).
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Fig. 2 Cross-section of neuropile glial cell in the region of its maximal extent. Two large medial processes run to afferent axons (aA); numerous small processes enclose axons (A) in the neuropile. The cytoplasm of the NPG cell is separated from connective tissue (CT) by numerous small microglial cells (arrows). There are also lipid drops (L), filaments (F) and endoplasmic reticulum (ER) in the peripheral zone. Electron microscopy (EM).
The third, outer zone within the central cytoplasm was characterized by extensive, parallel membrane stacks which were separated from each other by distances of up to 0.1-0.2 gm (Figs 2, 3). By staining with lanthanum nitrate and tannic acid as extracellular tracers, we tried to discern whether these stacks were composed of intracellular membranes or represented invaginations of the outer cell membrane. These membranes could not be contrasted by either of these reagents, while the outer surface of the NPG cell was distinctly contrasted. We conclude that these membrane stacks are part of an extensive smooth endoplasmic reticulum. The numerous processes of NPG cells in the neuropile can be distinguished from axons by their electron-dense cytoplasm (Figs 6, 8, 9) and by their lipid-drop content (Fig. 9). They surround axons or axon bundles as a network and lie close to them, separated only by small intercellular gaps (Figs 7, 8). NPG cell processes are also characterized by bundles of intermediate filaments (Fig. 8). These filaments are connected to the inner ganglion capsule by hemidesmosomes (Fig. 8, arrows). At the synaptic regions of the neuropile, the NPG processes almost surround axon bundles but only in a few cases were they observed near synapses, although never in direct contact (Fig. 7).
Detection of GFAP Intermediate filaments of invertebrate glial cells have not been reported to react with antibodies against vertebrate GFAP. We found a positive reaction in NPG cells of Hirudo with polyclonal antibodies against human GFAP (Fig. 11). The areas of intense fluorescence correspond to the regions where intermediate filaments were found to be abundant. Inside the ventral medial part of the neuropile there is a ring-shaped fluorescent site corresponding to the location of filaments inside the central cytoplasm of the NPG cell (Fig. 11, arrows). The NPG-cell processes in the neuropile were also distinctly marked (Fig. 11, arrowheads) and there was a strong fluorescence at the periphery of the neuropile where NPG cells are attached by many filaments at hemidesmosomes. Control sections were incubated in rabbit preimmune serum for the same time and the primary antibody was omitted. Monoclonal anti-GFAP Igs (Boehringer) showed no positive reaction in the tissue of Hirudo.
Discussion General morphology and function Our results on the fine structure of the NPG cell in the central nervous system of Hirudo medicinalis are in good
agreement with earlier investigations of the same preparation (Gray and Guillery, 1963; Coggeshall and Fawcett, 1964). However, it is difficult to compare these observations of NPG-cell ultrastructure with those of other invertebrate glial cells because of non-uniform use of methods and terminology, incomplete ultrastructural description and insufficient site descriptions in earlier investigations. Light microscopic examinations in other species of leech show the presence of glial cells differing in size and number from those in the medicinal leech (for review see Sawyer, 1986). Only ultrastmctural results from the horse leech (Haemopis sanguisuga) indicate a glial cell distribution similar to that in the medicinal leech (Kai-Kai and Pentreath, 1981). It is possible that the NPG cell processes ensheath neuronal axons and function as a 'loose myelin wrapping' (Coggeshall and Fawcett, 1964) as has been described for axon-wrapping glia of snails (Reinecke, 1975, 1976), and for insects (Pentreath, 1989). However, we consider this expression inappropriate here, because the presence of myelin basic protein in the NPG processes has not been confirmed. There are also no studies that indicate that these processes speed up neuronal impulse transmission, as is the case for the giant axons of the earthworm (Gtinther, 1976). In contrast to earlier studies (Coggeshall, 1974), we found no gap junctions between glial cells in the leech central nervous system. Kuffier and Potter (1964) postulated the existence of gap junctions between NPG and packet glial cells on the basis of electrophysiological experiments. Using fluorescent markers, evidence for gap junctions was found between the two NPG cells of a single ganglion in young specimens of Hirudo medicinalis (Schirrmacher and Deitmer, 1986), but these findings could not be confirmed in adult nervous systems of Hirudo (Schlue et al., 1980).
Endoplasmic reticulum and lipid content The existence of numerous different organelles in NPG cells cannot support the hypothesis that NPG cells contain a less active cytoplasm than packet glial cells (Coggeshall and Fawcett, 1964). The NPG cells of the medicinal leech show a significant zonation of their central cytoplasm, which has not been described previously for other invertebrate glial cells. The peripheral zone contains numerous membrane stacks that could not be contrasted by lanthane nitrate or tannic acid, extracellular markers which can be used to delineate membrane invaginations (Kai-Kai and Pentreath, 1981). We conclude, therefore, that these membrane stacks belong to an extensive smooth endoplasmic reticulum, which is characteristic of cells with active lipid metabolism. This conclusion would thus be consistent with our finding that NPG cells contain numerous large lipid drops and that their presence is related to nutrition (unpublished observations) and temperature (Spinedi et al., 1987).
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Fig. 3 Ultrathin section showing zonation of NPG-cell central cytoplasm. The inner region contains numerous ribosomes and mitochondria, glial filaments (F) and lipid drops (L). At the periphery lie stacks of membranes of smooth endoplasmic reticulum (ER). The NPG cell processes can be distinguished from axons (A) by their more electron-dense cytoplasm (upper figure detail). CT, connective tissue. EM.
Fig. 4 Detail of central cytoplasm in leech NPG cell. There'are ribosomes, dictyosomes (D), numerous mitochondria (M), various vesicles and large lipid drops. Nu, nucleus of microglial cell (MG). EM.
Synaptic region In the horse leech (Kai-Kai and Pentreath, 1981) the processes of NPG cells are very sparse, especially in synaptic regions, and it has been concluded that these NPG cells are not involved in synaptic transmission. In contrast, in the medicinal leech central nervous system we found numerous NPG cell processes near synapses. Electrophysiological investigations have shown that NPG cells contain receptors for acetylcholine (Ballanyi and Schlue, 1989) and glutamate (Ballanyi and Schlue, 1989; D6rner et al., 1990, 1994; Hochstrate and Schlue, 1994), and that they react to these neurotransmitters. NPG cells could play an important role at synapses in the same way as astrocytes are presumed to function (Roots, 1986; Kimelberg, 1987).
Glial intermediate filaments NPG cells contain bunches of cytoplasmic intermediate filaments and processes that correspond in thickness to glial filaments of vertebrates (Lazarides, 1982). These appear to be connected to hemidesmosomes, and could play an important role in stabilizing the tissue during mechanical stress which might arise when the animal changes its length. Intermediate filaments occur in many invertebrates with the exception of the Arthropoda, but until now they have not been characterized biochemically in invertebrates (Bartnik and Weber, 1989). In vertebrate astrocytes, GFAP is the main protein of gliai filaments (Eng and DeArmond, 1983) and it is used as a specific glia marker (Nanjoks-Manteuffel and Roth, 1989). These filaments are involved in neuronal migration during development, and interruption of GFAP synthesis in embryos produces injury in neurons (Renau-Piqueras et al., 1989). Damage to nervous tissue may also elevate GFAP content (Hall et al., 1989). Our experiments support the opinion that GFAP is also the main protein of leech glial cells. The sites of GFAP immunocytochemical detection correspond well to the sites of intermediate filaments in ultrathin sections. Because there can be different kinds of intermediate filaments in one cell (Naujoks-Manteuffel and Roth, 1989) we cannot exclude that other structural proteins (vimentin for example) are incorporated into glial filaments of Hirudo as in astrocytes of Mammalia (Lazarides, 1982) and Urodela (Zamora and Mutin, 1988).
Functional significance of NPG cell morphology The results of the present investigation and our previous findings on the localization of carbonic anhydrase in glial
cells of Hirudo medicinalis (Riehl and Schlue, 1990, 1993) suggest that NPG cells, according to their morphological properties and cytoplasmic contents, seem to have a high metabolic activity. Active lipid metabolism of NPG cells and their involvement in supplying neurons with energyrich metabolites (Pentreath, 1989) could be indicated by the extensive membrane stacks arranged in parallel within the central cytoplasm and by the high lipid content of their cell bodies and processes. Furthermore, the morphology of the NPG cells implies that they could be involved in a number of different physiological processes in the central nervous system of Hirudo. NPG cells shield axons from the hemolymph, and with a large surface in contact with the extracellular fluid, they may serve to regulate the ionic composition of the extracellular space. Investigations using electrophysiological methods to study the regulation of K* (Schlue and Wuttke, 1983; Walz et al., 1983) and H + ions (Deitmer and Schlue, 1987; Schlue et al., 1991) by NPG cells, and the effects of neurotransmitters on NPG cells (Walz and Schlue, 1982; Ballanyi and Schlue, 1989; D6rner et al., 1990) support the conclusion that NPG cells contribute to the regulation of the microenvironment surrounding neurons and glial cells. The distribution of glial filaments in NPG cells corresponds to the distribution of divalent cations in these cells. Investigations on glial filaments show that they bind Ca >, which activates enzymes for their decomposition and assembly (Yang and Babitch, 1988; Yang et al., 1988). It was concluded that Ca 2+ does have a structural and regulatory role in relation to glial filaments in astrocytes, and it may perform a similar function in Hirudo. Since in NPG cells membrane depolarization and neurotransmitters cause a significant increase in the intracellular free Ca 2÷ concentration (Deitmer and Schlue, 1987; Munsch and Deitmer, 1992; Hochstrate and Schlue, 1994), it appears possible that their filament system undergoes dynamic changes in response to neuronal activity. ACKNOWLEDGEMENTS This study was supported by DFG grants to W.-R. Schlue (Schl 169/8-2, Schl 169/12-1 and 12-2). We thank Dr Hartmut Greven for the supply of technical equipment, and Hanne Horn for excellent photographic assistance.
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Fig. 5 Detail of the periphery of a NPG cell with nucleus (lower figure half) in the neighbourhood of a packet glial cell (upper figure half). The cytoplasm of the NPG cell is separated from the packet glial cell and connective tissue (CT) by electron-dense microglial cells (MG). D: dictyosome; F, filaments of packet glial cell. EM. Fig. 6
Processes of a NPG cell in neuropile (arrows). The cytoplasm is more electron-dense than that of the axons (A). Process diameter differs. EM.
Fig. 7
Location of a synapse (arrow) in neuropile in relation to processes of NPG cell (arrowheads). A, axons; M, mitochondrion. EM.
MORPHOLOGICAL ORGANIZATION OF GLIAL CELLS IN THE LEECH NERVOUS SYSTEM
Fig. 8
Glial filaments (F) in processes of NPG cell between axons (A) are connected to hemidesmosomes (arrows). EM.
Fig. 9
Lipid drops (L) in electron-dense cytoplasm of NPG cell processes in the neuropile. A, axons; M, mitochondrion of axon. EM.
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Fig. 10 Cross-section detail of leech ganglion after staining with Sudan III (for neutral lipids and lipoids). In the cytoplasm of NPG cell lipid drops are arranged in a ring shape (arrows). N, neurons; NP, neuropile. Cryo section. LM. Fig. l l Cross-section of leech ganglion after immunocytochemical reaction against GFAP. In the ventral mid-portion of the neuropile (NP) there is a ring-shaped fluorescent structure (arrows) that corresponds to the arrangement of glial filaments in the cytoplasm of the NPG cell. There are numerous processes in the neuropile (arrowheads) that show a positive reaction. At the periphery, where many NPG cell filaments connect to hemidesmosomes, the fluorescence becomes more intense. Cryo section. LM.
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Morphological organization of neuropile glial cells in the central nervous system of the medicinal leech (Hirudo medicinalis) B. Riehl, W.-R. Schlue
Abstract Neuropile glial (NPG) cells in the central nervous system of the medicinal leech, Hirudo medicinalis, were studied by histological, histochemical and immunocytochemical techniques. The NPG cells are often surrounded by electron-dense microglial cells. The central cytoplasm of NPG cells shows a significant zonation. The zone around the nucleus contains mitochondria, glycogen and vesicles. The cytoplasm also contains many ribosomes, a few dictyosomes and distinct inclusions up to 2 gm in diameter. A second zone around the perinuclear region is marked by the occurrence of bundles of intermediate filaments that correspond in thickness to glial filaments of vertebrates. We found a positive reaction with polyclonal antibodies against human glial fibrillary acidic protein (GFAP), and the areas of intense fluorescence correspond to the regions where intermediate filaments were found to be abundant. The peripheral zone contains numerous membrane stacks that could not be contrasted by lanthane nitrate or tannic acid. Therefore, the membrane stacks could be part of an extensive smooth endoplasmic reticulum, which is characteristic of cells with active lipid metabolism. Keywords: Leech,glial filaments, GFAP, ultrastructure,lipid content, neuropileglial cell
Introduction Glial cells in central nervous tissues of invertebrates are found at the border between hemolymph or blood and neuronal elements, where they surround neurons and their processes. It is difficult to compare morphological studies of glial cells in different invertebrates, because so many different glial cell types have been described on the basis of experiments using various markers (Radojcic and Pentreath, 1979). No morphological classification has been undertaken in invertebrates, and there is need for the definition of reliable characteristics (Roots, 1986). Institut for Neurobiologie, Heinrich-Heine-Universit~R Desseldorf, Universit&tsstr. 1, D-40225 DLisseldorf, Germany. Received 29 April 1997 Accepted 13 November 1997 Correspondence to: Dr Wolf-R0diger Schlue. Tel: (49) (211) 81 13414; Fax: (49) (211 ) 81 13279; E-mail: schlue @uni-duesseldorf.de.
The central nervous system of Hirudo medicinalis has been a standard preparation for electrophysiological experiments, because of its uniform and simple organization with good access to individual glial cells and neurons. The location, morphology and physiology of the neurons have been studied extensively (for review see Sawyer, 1986). In recent years there has been an increasing interest in the properties and functions of glial cells in the leech central nervous system (for review see Pentreath, 1989); however, the histological characterization of this type of cell is incomplete at present. Macroglial cells of the leech have been previously identified according to their location using electron microscopy (Gray and Guillery, 1963; Coggeshall and Fawcett, 1964) but these studies did not investigate the ultrastructure of neuropile glial (NPG) cells. These NPG cells have served as a model system with regard to the involvement of glial cells in processes of K +, H + and Ca 2+ regulation (Schlue and Wuttke, 1983; Schlue and Walz, 1984; Schlue et al., 1991; 177
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Hochstrate and Schlue, 1994). The NPG cells have also provided a suitable model system for investigating the ways in which certain neurotransmitters, i.e. 5-hydroxytryptamine, acetylcholine and glutamate, interact with receptors in the glial membranes so as to control the homeostasis of ions in the nervous system (Walz and Schlue, 1982; Ballanyi and Schlue, 1989; Dtrner et al., 1990, 1994; Hochstrate and Schlue, 1994). The cell bodies of NPG cells are located in the ventral mid-region of each neuropile and their processes surround neuropile axons and synaptic regions. As demonstrated previously, NPG cells can be identified by their carbonic anhydrase II activity (Riehl and Schlue, 1990, 1993). In the present study we investigate the ultrastructural properties of NPG cells using conventional electron microscopy, to elucidate their significant relationship to the physiological function of these cells. We found no evidence for gap junctions between these cells but, according to their morphological properties and cytoplasmic contents, NPG cells could be involved in a number of different physiological processes. We show that a significant zonation is present in the central cytoplasm of NPG cells, and that the peripheral zone contains numerous membrane stacks which could not be contrasted by lanthane nitrate or tannic acid. Combining the conventional and fluorescence techniques, we also demonstrate that bundles of intermediate filaments in NPG cells contain glial fibrillary acidic protein (GFAP), which is characteristic of vertebrate astrocytes (Traub, 1985).
Materials and methods Tissue preparation for ultrastructural examination Adult medicinal leeches were sectioned and tissues were fixed in a mixture of 2% formaldehyde and 0.2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 1 h at 4°C. The chosen reagents allowed good preservation of tissue for light- and electron-microscope examination. We noted that concentrations of glutaraldehyde exceeding 0.2% damaged the ultrastructure of NPG cells, although the surrounding tissue was well preserved. It appeared that other mixture compositions did not reach NPG cells rapidly enough to prevent destruction of tissue. After washing with 0.1 M sodium cacodylate buffer, the tissue was postfixed in a solution of 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at 4°C. In addition we used postfixation solutions of 1% lanthane nitrate (Shaklai and Tavassoli, 1977) or 2% tannic acid (Futaesaku et al., 1972) as extracellular markers. After additional washing in 0.1 M sodium cacodylate buffer tissues were dehydrated in an ascending series of acetone and embedded in TAAB epoxide (Araldite). Semithin sections, 0.3-0.5%tm thick, were cut on a Reichert Ultracut and stained with an aqueous solution of 1% methylene blue/l% disodium tetraborate, 1:1. Silvergrey ultrathin sections were cut with a diamond knife, stretched by xylene and collected on pioloform (Merck)
coated copper grids. Sections were contrasted for 30 min in a methanolic solution of uranyl acetate, washed with distilled water and dried. Next we incubated preparations for 1 min in lead-II-citrate-trihydrate (Venable and Coggeshall, 1965). Photographs were made with a Zeiss EM 9S electron microscope and an Olympus BH-2 light microscope.
Histochemical detection of lipid content Frozen tissue sections were briefly dehydrated and covered by a 50% ethanol solution for 30 s. To stain for neutral lipids and lipoids, sections were dropped into a Sudan III solution according to Daddi (cf. Adam and Czihak, 1964) for 30-60 s at room temperature. After another 30 s in 50% ethanol and 2 min rinsing in distilled water, sections were mounted with glycerol-gelatine. For distinction between acidic and neutral lipids, sections were incubated in 1% aqueous Nile Blue sulfate at 50°C for 5 min, rinsed in distilled water at 60°C and stained again in 0.2% Nile Blue sulfate solution. After rinsing in distilled water, sections were prepared as described above. Immunocytochemical detection of GFAP Tissue was examined by the indirect immunofluorescence technique with FITC-coupled antiserum according to Sternberger (1979). Non-specific labeling was blocked by a 30-min treatment of sections with normal goat serum (1:50 in PBS at room temperature). This step was followed by incubation with anti-human GFAP serum from rabbits (Dakopatts) in solutions between 1:50 and 1:500 in PBS for 1 h at room temperature or overnight at 4°C. After washing three times in PBS buffer for 10 min at 4°C, the slides were incubated for 1 h at room temperature with goat anti-rabbit Ig coupled with FITC (Sigma) diluted 1:50 in PBS. After additional washing in PBS the sections were viewed and photographed in an Olympus BH2 fluorescence microscope using excitation filters EY-455 and BP-490, dichroic mirror 500 and barrier filter 0-515.
Results Morphology and lipid content of NPG cells A single leech ganglion is delimited by an outer capsule (Fig. 1). There is also an inner ganglion capsule enclosing axons and cell bodies, occasional neuron somata in the periphery of the neuropile, many microglial cells scattered through the neuropile, and two especially large glial cells lying next to the neuropile in the anterior and posterior regions of each ganglion, the NPG cells. Analysis of serial sections showed that the central cytoplasm of the NPG cells is located at the central midline in each ganglion. The somata of the cells send out projections that enter the neuropile and branch within it; here they could interact with the synapses. This morphological characteristic in itself makes the NPG cells an interesting candidate for studies of neuron-glia interactions, for in the leech (as in other invertebrates) the fundamental operations of the
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Fig. 1 Cross-section through a ganglion of Hirudo medicinalis. There is an outer and inner capsule of connective tissue (CT) dividing the region of packet glial cells (PG) and neurons (N) from the inner neuropile (NP) and from the neuropite glial cell (NPG). The neuropile contains axons (A) and four synaptic regions (s). Inside the cytoplasm of the neuropile glial cell are numerous lipid drops (arrows), which are also in the processes (arrowheads). Semithin section (Araldite, Azur II/Borax). Light microscopy (LM).
central nervous system, that is, synaptic transmission and integration, occur in the neuropile. There are four main processes of these cells, in addition to many small processes extending from the cell body of the NPG cell into the neuropile (Fig. 2). Two of the large processes run laterally parallel to the inner connective tissue and completely enclose the mass of axons in the neuropile. The two others lie medially, dorsal to the central NPG cytoplasm, and are close to the 'giant afferent neurons' in the central neuropile (Fernandez, 1978). We did not find any direct contacts between NPG and other macroglial cells. At the regions where the neuronal processes cross the inner capsule, they are surrounded outside the neuropile by the packet glial cells and inside by the NPG cells. The two glial cell types are separated from one another by connective tissue or by microglial cells (Fig. 5). The NPG cells are often surrounded by electron-dense microglial cells and their processes (Figs 2, 5). Like all macroglial cells of the leech, NPG cells have a nucleus
with dense, filamentous material at its outer envelope (Fig. 5). The nucleoplasm is light, with a similar appearance to the neuronal nuclei. The cytoplasm of NPG cells is more electron-dense than that of other leech macroglial cells, and shows a marked division into zones. In the zone around the nucleus mitochondria, glycogen and vesicles of different content and size are regularly distributed. The cytoplasm also contains many ribosomes and a few dictyosomes (Fig. 4). Even in light-microscope sections distinct inclusions with a diameter up to 2 gm can be seen (Fig. 1). In electron micrographs these inclusions did not appear to be limited by a membrane and their abundance depended on the nutritional status of the animals. Staining with Sudan III (Fig. 10) and Nile Blue sulfate confirmed that the inclusions are lipids within the NPG cytoplasm. We found these lipids to be of acidic and neutral types. A second zone around the perinuclear zone described above was marked by the occurrence of many small bundles of intermediate filaments, in an annular arrangement (Fig. 3).
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Fig. 2 Cross-section of neuropile glial cell in the region of its maximal extent. Two large medial processes run to afferent axons (aA); numerous small processes enclose axons (A) in the neuropile. The cytoplasm of the NPG cell is separated from connective tissue (CT) by numerous small microglial cells (arrows). There are also lipid drops (L), filaments (F) and endoplasmic reticulum (ER) in the peripheral zone. Electron microscopy (EM).
The third, outer zone within the central cytoplasm was characterized by extensive, parallel membrane stacks which were separated from each other by distances of up to 0.1-0.2 gm (Figs 2, 3). By staining with lanthanum nitrate and tannic acid as extracellular tracers, we tried to discern whether these stacks were composed of intracellular membranes or represented invaginations of the outer cell membrane. These membranes could not be contrasted by either of these reagents, while the outer surface of the NPG cell was distinctly contrasted. We conclude that these membrane stacks are part of an extensive smooth endoplasmic reticulum. The numerous processes of NPG cells in the neuropile can be distinguished from axons by their electron-dense cytoplasm (Figs 6, 8, 9) and by their lipid-drop content (Fig. 9). They surround axons or axon bundles as a network and lie close to them, separated only by small intercellular gaps (Figs 7, 8). NPG cell processes are also characterized by bundles of intermediate filaments (Fig. 8). These filaments are connected to the inner ganglion capsule by hemidesmosomes (Fig. 8, arrows). At the synaptic regions of the neuropile, the NPG processes almost surround axon bundles but only in a few cases were they observed near synapses, although never in direct contact (Fig. 7).
Detection of GFAP Intermediate filaments of invertebrate glial cells have not been reported to react with antibodies against vertebrate GFAP. We found a positive reaction in NPG cells of Hirudo with polyclonal antibodies against human GFAP (Fig. 11). The areas of intense fluorescence correspond to the regions where intermediate filaments were found to be abundant. Inside the ventral medial part of the neuropile there is a ring-shaped fluorescent site corresponding to the location of filaments inside the central cytoplasm of the NPG cell (Fig. 11, arrows). The NPG-cell processes in the neuropile were also distinctly marked (Fig. 11, arrowheads) and there was a strong fluorescence at the periphery of the neuropile where NPG cells are attached by many filaments at hemidesmosomes. Control sections were incubated in rabbit preimmune serum for the same time and the primary antibody was omitted. Monoclonal anti-GFAP Igs (Boehringer) showed no positive reaction in the tissue of Hirudo.
Discussion General morphology and function Our results on the fine structure of the NPG cell in the central nervous system of Hirudo medicinalis are in good
agreement with earlier investigations of the same preparation (Gray and Guillery, 1963; Coggeshall and Fawcett, 1964). However, it is difficult to compare these observations of NPG-cell ultrastructure with those of other invertebrate glial cells because of non-uniform use of methods and terminology, incomplete ultrastructural description and insufficient site descriptions in earlier investigations. Light microscopic examinations in other species of leech show the presence of glial cells differing in size and number from those in the medicinal leech (for review see Sawyer, 1986). Only ultrastmctural results from the horse leech (Haemopis sanguisuga) indicate a glial cell distribution similar to that in the medicinal leech (Kai-Kai and Pentreath, 1981). It is possible that the NPG cell processes ensheath neuronal axons and function as a 'loose myelin wrapping' (Coggeshall and Fawcett, 1964) as has been described for axon-wrapping glia of snails (Reinecke, 1975, 1976), and for insects (Pentreath, 1989). However, we consider this expression inappropriate here, because the presence of myelin basic protein in the NPG processes has not been confirmed. There are also no studies that indicate that these processes speed up neuronal impulse transmission, as is the case for the giant axons of the earthworm (Gtinther, 1976). In contrast to earlier studies (Coggeshall, 1974), we found no gap junctions between glial cells in the leech central nervous system. Kuffier and Potter (1964) postulated the existence of gap junctions between NPG and packet glial cells on the basis of electrophysiological experiments. Using fluorescent markers, evidence for gap junctions was found between the two NPG cells of a single ganglion in young specimens of Hirudo medicinalis (Schirrmacher and Deitmer, 1986), but these findings could not be confirmed in adult nervous systems of Hirudo (Schlue et al., 1980).
Endoplasmic reticulum and lipid content The existence of numerous different organelles in NPG cells cannot support the hypothesis that NPG cells contain a less active cytoplasm than packet glial cells (Coggeshall and Fawcett, 1964). The NPG cells of the medicinal leech show a significant zonation of their central cytoplasm, which has not been described previously for other invertebrate glial cells. The peripheral zone contains numerous membrane stacks that could not be contrasted by lanthane nitrate or tannic acid, extracellular markers which can be used to delineate membrane invaginations (Kai-Kai and Pentreath, 1981). We conclude, therefore, that these membrane stacks belong to an extensive smooth endoplasmic reticulum, which is characteristic of cells with active lipid metabolism. This conclusion would thus be consistent with our finding that NPG cells contain numerous large lipid drops and that their presence is related to nutrition (unpublished observations) and temperature (Spinedi et al., 1987).
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Fig. 3 Ultrathin section showing zonation of NPG-cell central cytoplasm. The inner region contains numerous ribosomes and mitochondria, glial filaments (F) and lipid drops (L). At the periphery lie stacks of membranes of smooth endoplasmic reticulum (ER). The NPG cell processes can be distinguished from axons (A) by their more electron-dense cytoplasm (upper figure detail). CT, connective tissue. EM.
Fig. 4 Detail of central cytoplasm in leech NPG cell. There'are ribosomes, dictyosomes (D), numerous mitochondria (M), various vesicles and large lipid drops. Nu, nucleus of microglial cell (MG). EM.
Synaptic region In the horse leech (Kai-Kai and Pentreath, 1981) the processes of NPG cells are very sparse, especially in synaptic regions, and it has been concluded that these NPG cells are not involved in synaptic transmission. In contrast, in the medicinal leech central nervous system we found numerous NPG cell processes near synapses. Electrophysiological investigations have shown that NPG cells contain receptors for acetylcholine (Ballanyi and Schlue, 1989) and glutamate (Ballanyi and Schlue, 1989; D6rner et al., 1990, 1994; Hochstrate and Schlue, 1994), and that they react to these neurotransmitters. NPG cells could play an important role at synapses in the same way as astrocytes are presumed to function (Roots, 1986; Kimelberg, 1987).
Glial intermediate filaments NPG cells contain bunches of cytoplasmic intermediate filaments and processes that correspond in thickness to glial filaments of vertebrates (Lazarides, 1982). These appear to be connected to hemidesmosomes, and could play an important role in stabilizing the tissue during mechanical stress which might arise when the animal changes its length. Intermediate filaments occur in many invertebrates with the exception of the Arthropoda, but until now they have not been characterized biochemically in invertebrates (Bartnik and Weber, 1989). In vertebrate astrocytes, GFAP is the main protein of gliai filaments (Eng and DeArmond, 1983) and it is used as a specific glia marker (Nanjoks-Manteuffel and Roth, 1989). These filaments are involved in neuronal migration during development, and interruption of GFAP synthesis in embryos produces injury in neurons (Renau-Piqueras et al., 1989). Damage to nervous tissue may also elevate GFAP content (Hall et al., 1989). Our experiments support the opinion that GFAP is also the main protein of leech glial cells. The sites of GFAP immunocytochemical detection correspond well to the sites of intermediate filaments in ultrathin sections. Because there can be different kinds of intermediate filaments in one cell (Naujoks-Manteuffel and Roth, 1989) we cannot exclude that other structural proteins (vimentin for example) are incorporated into glial filaments of Hirudo as in astrocytes of Mammalia (Lazarides, 1982) and Urodela (Zamora and Mutin, 1988).
Functional significance of NPG cell morphology The results of the present investigation and our previous findings on the localization of carbonic anhydrase in glial
cells of Hirudo medicinalis (Riehl and Schlue, 1990, 1993) suggest that NPG cells, according to their morphological properties and cytoplasmic contents, seem to have a high metabolic activity. Active lipid metabolism of NPG cells and their involvement in supplying neurons with energyrich metabolites (Pentreath, 1989) could be indicated by the extensive membrane stacks arranged in parallel within the central cytoplasm and by the high lipid content of their cell bodies and processes. Furthermore, the morphology of the NPG cells implies that they could be involved in a number of different physiological processes in the central nervous system of Hirudo. NPG cells shield axons from the hemolymph, and with a large surface in contact with the extracellular fluid, they may serve to regulate the ionic composition of the extracellular space. Investigations using electrophysiological methods to study the regulation of K* (Schlue and Wuttke, 1983; Walz et al., 1983) and H + ions (Deitmer and Schlue, 1987; Schlue et al., 1991) by NPG cells, and the effects of neurotransmitters on NPG cells (Walz and Schlue, 1982; Ballanyi and Schlue, 1989; D6rner et al., 1990) support the conclusion that NPG cells contribute to the regulation of the microenvironment surrounding neurons and glial cells. The distribution of glial filaments in NPG cells corresponds to the distribution of divalent cations in these cells. Investigations on glial filaments show that they bind Ca >, which activates enzymes for their decomposition and assembly (Yang and Babitch, 1988; Yang et al., 1988). It was concluded that Ca 2+ does have a structural and regulatory role in relation to glial filaments in astrocytes, and it may perform a similar function in Hirudo. Since in NPG cells membrane depolarization and neurotransmitters cause a significant increase in the intracellular free Ca 2÷ concentration (Deitmer and Schlue, 1987; Munsch and Deitmer, 1992; Hochstrate and Schlue, 1994), it appears possible that their filament system undergoes dynamic changes in response to neuronal activity. ACKNOWLEDGEMENTS This study was supported by DFG grants to W.-R. Schlue (Schl 169/8-2, Schl 169/12-1 and 12-2). We thank Dr Hartmut Greven for the supply of technical equipment, and Hanne Horn for excellent photographic assistance.
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Fig. 5 Detail of the periphery of a NPG cell with nucleus (lower figure half) in the neighbourhood of a packet glial cell (upper figure half). The cytoplasm of the NPG cell is separated from the packet glial cell and connective tissue (CT) by electron-dense microglial cells (MG). D: dictyosome; F, filaments of packet glial cell. EM. Fig. 6
Processes of a NPG cell in neuropile (arrows). The cytoplasm is more electron-dense than that of the axons (A). Process diameter differs. EM.
Fig. 7
Location of a synapse (arrow) in neuropile in relation to processes of NPG cell (arrowheads). A, axons; M, mitochondrion. EM.
MORPHOLOGICAL ORGANIZATION OF GLIAL CELLS IN THE LEECH NERVOUS SYSTEM
Fig. 8
Glial filaments (F) in processes of NPG cell between axons (A) are connected to hemidesmosomes (arrows). EM.
Fig. 9
Lipid drops (L) in electron-dense cytoplasm of NPG cell processes in the neuropile. A, axons; M, mitochondrion of axon. EM.
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Fig. 10 Cross-section detail of leech ganglion after staining with Sudan III (for neutral lipids and lipoids). In the cytoplasm of NPG cell lipid drops are arranged in a ring shape (arrows). N, neurons; NP, neuropile. Cryo section. LM. Fig. l l Cross-section of leech ganglion after immunocytochemical reaction against GFAP. In the ventral mid-portion of the neuropile (NP) there is a ring-shaped fluorescent structure (arrows) that corresponds to the arrangement of glial filaments in the cytoplasm of the NPG cell. There are numerous processes in the neuropile (arrowheads) that show a positive reaction. At the periphery, where many NPG cell filaments connect to hemidesmosomes, the fluorescence becomes more intense. Cryo section. LM.
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