Ultrastructural Identification of Glial Cells in the Oral Area of the Comb-Bearer Beroё Cucumis M

Home , Mucus

Journal of Evolutionary Biochemistry and Physiology, Vol. 40, No. 6, 2004, pp. 710—720. Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 40, No. 6, 2004, pp. 579—588. Original Russian Text Copyright © 2004 by Aronova, Alekseeva.

To the 100-Anniversary of A. K. Voskresenskaya

Ultrastructural Identification of Glial Cells in the Oral Area of the Comb-Bearer Beroё cucumis M. Z. Aronova and T. M. Alekseeva Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia Received July 4, 2003

Abstract—In the electron microscopic study of oral epithelium and underlying areas of mesoglea of adult comb-bearers Beroё cucumis (Ctenophora), there were described peculiar light cells with a characteristic localization, ultrastructure, ways of interaction with cells of other types including developing and mature chemoreceptors and neuronal processes. Comparison of the obtained results with the literature data allows identification of the light cells as glial cells, one of the first such cells in representatives of the first multicellular animals.

INTRODUCTION also be associated with their participation in utili- zation of degenerated chemoreceptors. In an electron microscopic study of mouth epi- This paper presents results of electron micro- thelium of adult comb-bearers Beroё cucumis scopic study of the light cells. Comparison of data (Ctenophora) we have described consecutive stages on their localization and ultrastructural organiza- of chemoreceptor cell maturation [1]. On enter- tion with known morphological characteristics of ing the epithelium from mesoglea the precursor glial elements of the higher multicellular animals cells are transformed into juvenile and then into can help elucidation of the nature of these myste- mature chemoreceptor elements. This results in rious cells revealed on the oral pole of the comb- replacement of damaged and destroyed cells by bearers Beroё cucumis. new cells. Penetration of precursor cells into epithelium was preceded by damage of the basement MATERIALS AND METHODS membrane and the appearance of its defects. Near these sites, peculiar light cells were found, which In 15 adult comb-bearers Beroё cucumis (Cteno- were comparable with glial cells by several features phora) kept in sea water, fragments of oral epithe- [1]. Preliminary data indicated possible interaction lium with underlying mesoglea were dissected out. of these cells with developing chemoreceptors and The material was first prefixed in 2–6% glutaral- their role in transfer of precursor elements and dehyde in cacodylate buffer, pH 7.2, at 10°C for nerve endings from mesoglea into the chemorecep- 1–2 h, then, after a quick washing in the same buff- tor epithelium. At the same time, the presence of er, placed in cold 1% OsO4. Contrasting of the fixed phagocyted cell fragments in the cytoplasm can objects with a mixture of 0.5% uranyl acetate and

1% phosphotungstic acid in 70° ethanol was car- both pre- and postsynaptic areas (Fig. 1e). Of spe- ried out in the course of their dehydration. After cial interest was the presence in these sites of me- that, they were embedded in Epon, cut in an LKB soglea and in adjacent parts of receptor epitheli- 812 Ultratome, and examined in JEM 100B, JEM um of cells with cytoplasm of low electron density; 1200EX, and LEO-910 translucent electron mi- we designated them as light cells [1]. Perikarya of croscopes. Besides, there was used the previously these large, relatively not numerous elements had processed experimental material fixed for electron no constant shape, but more often were elongat- microscopic cytochemical study of Beroё epithe- ed, with large cytoplasmic outgrowths, one of lium. This explains the presence of granules of de- them, as a rule, being narrower and longer. No ar- posit appeared in some of presented electron mi- borization of the branches was revealed. In the croscopic photographs. loose cytoplasm with relatively few organelles there were found occasional mitochondria with light RESULTS AND DISCUSSION matrix and a few tubular cristae, moderately de- veloped structures of Golgi apparatus, microtu- Comb-bearers are among the first known mul- bules, rough endoplasmic membranes, lysosomal ticellular animals inhabiting sea water and moving bodies, and small accumulations of osmiophilic actively there with a mouth forth. Resistance of material. The chromatin-filled, bright, large nu- water masses, which is particularly significant dur- clei had, as a rule, triangular shape and centrally ing the high and low tide, and contact with various placed, electron dense, round nucleolus (Fig. 2a). obstacles can lead to a damage of chemoreceptor Synapses between nerve terminals and light cells, cells that are localized at the edge of mouth and regardless of location of these cells—whether in protected from the surface only by layers of glyco- epithelium or in mesoglea—had organization of the calix and mucus. The damaged cells are destroyed, type of synaptic triads, which are characteristic of removed from epithelium, and replaced by new comb-bearers (Figs. 2b–2d). Rather deep invagi- receptor elements. Previously we have found [1] nations of nerve endings into the body of the cyto- that some special light cells participate in the cycle plasmic outgrowth were revealed, predominantly of chemoreceptor development, including its ini- within the limits of the receptor epithelium tial steps associated with reorganization of precur- (Fig. 2e). There was some similarity of the light sor elements. The structure of these cells will be cells with the mesogleal ones. However, in the lat- considered in the present work. ter, also light and with processes and most likely Chemoreceptor cells in the oral epithelium of capable for locomotion and phagocytosis, the cy- Beroё cucumis are known to be distributed non- toplasm was more electron dense. It contained both uniformly—either singly or in groups of several individual microfilaments and their bundles. cells. In the groups there was observed a thicken- Sometimes mitochondria were concentrated in the ing of epithelial layer, whose lower third contained site of transition of the cytoplasm into a thin elec- basal areas of chemoreceptors, mucus-secreting tron dense, clearly seen process containing mi- cells, and juvenile and basal cells. Also located here crofilaments. Nuclei of mesogleal cells, either was a neuropil-like (Fig. 1a) accumulation of pro- elongated or of irregular shape, had a moderate cesses of nerve and receptor cells [1]. Mesoglea electron density. underlying these areas also was found to contain a Light cells were present in various areas of me- similar structure (Fig. 1b). Besides, some process- soglea, but more often in the sites of penetration es of nerve cells and central processes of receptor of precursor cells into the chemoreceptor epithe- cells, the mesogleal, smooth muscular and precur- lium; it is there that we have described them. Pe- sor cells, and interneurons were seen (Figs. 1c, 1d). culiarities of localization of these cells according The cells whose contents indicate their probable to electron microscopic photographs confirmed neurosecretory activity were remarkable. They the suggestion about their interaction with base- were solidly packed with osmiophilic vesicles and ment membrane [1]. Dissolved sites, defects, ap- dark granules and had synapses with nerve endings peared where the light cells were adjacent to the (Figs. 1d, 1e). Synaptic vesicles were clustered in basement membrane. In several sections, the light

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004 712 ARONOVA, ALEKSEEVA

Fig. 1. Cellular composition of basal areas of the chemoreceptor epithelium and underlying mesoglea of the comb- bearer. (a) Accumulation of neuropil-type elements (N ) in epithelium; (b) the same in mesoglea (Me), (c) interneurons (IN ) in Me, (d), (e) cell processes with osmiophilic vesicles (COV ), synapses (S ), PC—precursor cells, LC—light cell, LCP—process of the light cell, BM—basal membrane, Nu—nucleus, Nul—nucleolus, BBB—basal body and a basket of interneuron rootlet, MSC—basal area of mucus-secreting cells, M—mitochondria, CC—basal area of chemoreceptor cell. Magnification: (a), (b), (c) 20 000, (d) 23 000, (e) 40 000.

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004 ULTRASTRUCTURAL IDENTIFICATION OF GLIAL CELLS 713

Fig. 1. (Contd.). cells or their processes as well as precursor cells their ontogenesis they were transformed into ma- were already seen in epithelium. Subsequently, ju- ture chemoreceptors. These, in turn, replaced the venile cells also were found here; in the course of damaged degenerated chemoreceptors on moving

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004 714 ARONOVA, ALEKSEEVA

Fig. 2. Ultrastructure of light cells (LC) and synapses (S). NT—Nerve terminals, ST—synaptic triad, L—lysosomal bodies. Other designations as those in Fig. 1. Magnification: (a) 25 000, (b) 20 000, (c) 30 000, (d) 20 000.

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004 ULTRASTRUCTURAL IDENTIFICATION OF GLIAL CELLS 715

Fig. 2. (Contd.). to the surface of receptor epithelium [1]. The light bulb base can be surrounded only by the cytoplasm cells also take up and transport nerve endings to outgrowths of the Schwann or supporting cells in- epithelium (Figs. 3a, 3b). Electron microscopic stead of myelin sheaths [2]. pictures indicated a possibility of interaction of the Earlier, when discussing structure of the nervous light cells with the basal cells localized in epitheli- system of comb-bearers, we noted the presence in um [1], as they could be closely adjacent to the lat- it of cells, interneurons [3]. They were revealed ter (Fig. 3c). With time, integrity of the basement most often in epithelium and related sites of me- membrane was restored. soglea of the comb plate and chemoreceptor epi- As mentioned above, the light cell cytoplasm in thelium and much more seldom in the aboral sense some cases contained inclusions similar with frag- organ. Perikaryon of interneurons looked quite ments of destroyed chemoreceptors [1]. flattened, the nucleus was large, of irregular shape As the cytoplasmic outgrowths of mesogleal light and often multiblade, the nucleolus was dark and cells had moved away from the perikaryon, they located in one of the blades (Figs. 1c, 5a, 5d, and thinned to 200–100 nm and their electron density 5f ). In the Beroё interneurons, a long and proba- rose. Figure 4 shows central processes of chemore- bly highly coiled process can be guessed, its some ceptors running inside mesoglea and outside epi- areas being revealed either near the cell body thelium; they are surrounded by thin outgrowths (Fig. 5f ) or at a significant distance from it. In the of light cells [1]. The growth cone of the central cell body, under the plasma membrane, in the nar- process it remained free, not covered with these row space of perikaryon, a structure resembling a processes. According to our observations, the out- modified basal body was present, with a basket-like growths with a thin layer of the cytoplasm could rootlet, like the mature comb-bearer chemorecep- encompass not only individual central processes of tor (Figs. 1c, 5d–5f ). However, we never observed chemoreceptors, but also groups of several process- it going out onto the cell surface, like a flagellum es (Fig. 4). In this connection, of interest are data of chemoreceptor. No features characteristic of a on innervation of the taste bulb of vertebrates, ac- flagellum or a kinocilium could be detected in it, cording to which the nerve fibers coming to the but the circular arrangement of microfilament bun-

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004 716 ARONOVA, ALEKSEEVA

Fig. 3. Light cells and nerve terminals in the chemoreceptor epithelium. BC—Basal cells, PCC—processes of chemoreceptor cells. Other designations as those in Fig. 1. Magnification: (a) 30 000, (b) 25 000, (c) 16 000.

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004 ULTRASTRUCTURAL IDENTIFICATION OF GLIAL CELLS 717 dles leaving the rootlet basket could be compared with localization of peripheral fibrils of flagellar axoneme (Fig. 5d, 5e). At the same time, some similarity was revealed between its cross-striated fibrils (Fig. 5c) and nematocyst, the ejecting capsule of nematocytes in Coelenterates [3]. Structural evi- dence for its possible ejecting functions, like in kinocilium of Cnidaria, also was absent. It is to be particularly emphasized that interneurons of Beroё cucumis similar to all elements of locomotor-sensory system of comb-bearers, which we described earlier,—gravitational mechanoreceptor, photoreceptor, locomotor cells (cells of the comb plate), and smooth muscular cells [3, 4] are flagellate cells by their nature, which is indicated by the presence of rudimentary cilia (Fig. 5a, 5b). Numerous microtubules of apical perikaryon of interneurons in Beroё were distinguished by local- Fig. 4. Central processes of chemoreceptor cells ization, structure, and size. Rows of long microtu- (CPCC). SMC—Smooth muscle cell. Arrows—thin out- bules (Fig. 5f ) were arranged in parallel to the cell growths of light cells. Magnification: 13 000. Other designations as those in Fig. 1. surface and to each other, radially from walls of the cylinder formed by microfilament bundles (Figs. 5d, 5e). The deeper layers of the cytoplasm around the rootlet basket up to the border with existence in comb-bearers of reciprocal innerva- nucleus were filled with branching microtubules of tion that is absent in Coelenterata, but present in the larger diameter (Figs. 5a, 5d, 5f ). worms, molluscs, crustacean, and Echinoderma- Interneurons of comb-bearer, due to their loca- ta [5]. This agrees with our data about innervation tion, have no contacts with environment, but are of gravitational mechanoreceptor cells located in connected via synapses with corresponding effec- the aboral sense organ of Beroё. Morphological tors—the receptor cells of various modalities as well differences of nerve endings adjacent to basal ar- as with smooth muscle cells [3]. Peculiarities of eas of the cells have allowed us to suggest the exist- localization and ultrastructure of interneurons of ence of two types of innervation, efferent and af- Beroё cucumis permitted considering these cells as ferent, in comb-bearers. Some of them were tightly having motility and, besides, suggested their phy- packed with synaptic vesicles and looked dark, logenetic connection with both chemoreceptor comparable with efferent terminals. Others, on the and ancestor mobile nematocyte-like elements. In contrary, had only occasional light synaptic vesi- evolutionary line, interneurons are known to func- cles and were characterized as light nerve endings tionally have a progressive development by pro- presumably belonging to the afferent one [3]. cessing information of various kinds and sending In the aboral ganglion, apart from unipolar neu- signals to CNS. During this process the basic type rons, we described rounded cells with the large nu- of information collected from some particular cel- cleus, osmiophilic nucleolus, and cytoplasm of lular population is chosen. Thus, the scheme of moderate electron density as possible auxiliary glial organization of the Beroё nervous system reveals a elements [3]. There was revealed invagination of tendency for concentrating not only nerve ele- neuronal processes into the cytoplasm of these cells, ments with their processes in aboral ganglion, but which reminded the pattern of mesaxonal interac- also interneurons connecting effectors of different tions of nerve and glial elements of vertebrates. Near modalities with CNS [3]. the aboral ganglion in mesoglea as well as under D.M. Fedotov [5] attached the “doubtless evo- chemoreceptor epithelium, cellular processes filled lutionary and phylogenetic significance” to the with osmiophilic granules were observed.

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004 718 ARONOVA, ALEKSEEVA

Fig. 5. Interneurons in mesoglea of comb-bearers. (a) Perikaryon of interneuron with a rudimentary cilium (RC ); (b)— RC; (c), (d), (e) rootlet structures (RS); (f ) perikaryon and processes (Pr) of interneuron; BMf—bundles of microfilaments leaving the basal body; L—lines of parallel microtubules; AMT—microtubules with arborization. Other designations as those in Fig. 1. Magnification: (a) 30 000, (b) 40 000, (c) 43 000, (d) 40 000, (e) 50 000, (f ) 25 000.

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004 ULTRASTRUCTURAL IDENTIFICATION OF GLIAL CELLS 719

Glial cells of various kinds (including microglia, ization of light cell participation in control of bio- glial microphages) are known to affect developing chemical processes in the growth cone zone of the neurons, which is ascribed, in particular, to their chemoreceptor cell axon. ability to synthesize neurotrophic, neural growth The presence of synapses between light cells and factors [6] that control motility and growth of ax- nerve endings, which we described in Beroё, agrees ons. The control is performed via receptors present with recent data about “functional synapses” in on growth cones of neurons [7]. An example of ef- vertebrates as a form of interaction of neurons and fects of neurotrophic factors in the damaged stria- glial cells [12]. tum is a morphologically established increase of Structurally, the light cells, in our opinion, are arborization of neighboring dopaminergic fibers comparable with the glial elements belonging to [8]. Also known is role of glia in direction of neu- one of three main types of glial cells—with mi- roblast migration. There are data about participa- croglia or, more likely, with its structurally close tion of glia in regeneration of lesioned neurons, form. It is commonly believed that microglial cells retinal cells in tritons and lizards [9]. The polyfunc- are capable for ameboid movements and, due to tionality of glia is also seen in invertebrates due to active migration, are mostly spread throughout its role of a mechanical supporting structure, par- the whole nervous system. However, a part of ticipation in transport and accumulation of metab- them are located predominantly near neurons, olites, release and synthesis of neurotransmitters, which agrees with data about neurotrophic effects etc. [10, 11]. of glia on developing neurons. Classical micro- When describing light cells located near the glial cells (mesoglia, glial macrophages) have large mouth of Beroё cucumis, we noted some peculiar- size, shape of elongated processes, low electron ities of their ultrastructure and localization, which, density and contain few organelles and practical- in our opinion, can be considered as criteria for ly no filaments. Chromatin-rich nuclei of elon- elucidation of the nature of these cells. Light cells gated or triangular shape look dark against the both in mesoglea and chemoreceptor epithelium background of the light loose cytoplasm of the of Beroё were located near clusters of elements re- relatively large volume. Microglial cells are deriv- minding neuropil and including central processes atives of mesenchyma, unlike cells of macroglia of mature chemoreceptors, nerve endings, and (astrocytes, oligodendrocytes) originating from processes of interneurons. Some signs of phagocy- ectoderm. During lesion or degeneration, these tosis by light cells of debris of damaged and disin- cells proliferate, migrate to the focus, and perform tegrated cells were noted. Some fragments of os- phagocytic functions by transformation into mac- miophilic substance found in the cytoplasm of light rophages taking up products of degeneration of cells had shape and structure comparable morpho- other cells. In this case, glial macropages can ac- logically with glycogen granules or lipid inclusions. quire a rounded shape. The obtained morphological data indicated the The important role of glia is known to consist in capability of light cells for translocation and inter- formation of special sheaths around axons. In the action with other cells, particularly, chemorecep- simplest case, both in vertebrates and invertebrates, tor cells, at various stages of their morphogenesis— the single axon or a group of axons are immersed both developing and mature cells. In this connec- into the glial cell. Thereby, either the single non- tion, we pay a particular attention to the morpho- myelinated glial sheath or non-myelinated loose logically established fact that thin outgrowths of glial folds can be formed. However, no typical light cells are closely adjacent to the chemorecep- myelin sheath is formed, as a thin layer of cyto- tor central processes leaving epithelium and enter- plasm always remains inside the coil. ing mesoglea [1]. We consider this structural phe- The performed electron microscopic study of nomenon as the first attempts of the lower multi- structure and localization of light cells revealed cellular animals to form an additional sheath near the oral epithelium of the comb-bearers Beroё around the central process or group of the processes cucumis as well as a comparison with literature data to provide the quality of signal propagation along have allowed us to establish in these cells some sim- axon. Besides, this adjacency could result in real- ilarity with microglial cells, which takes place in

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004 720 ARONOVA, ALEKSEEVA the higher invertebrates as well as in vertebrate REFERENCES animals. As determining and basic signs and prop- erties characteristic of the light cells, we consid- 1. Aronova, M.Z. and Alekseeva, T.M., Development ered localization, shape, structure of the cytoplasm of Chemoreceptor Cells in Oral Epithelium of the and nucleus, and interaction with other cells. Adult Comb-Bearer Beroё cucumis, Zh. Evol. Bio- khim. Fiziol., 2003, vol. 39, pp. 578–586. As mentioned above, the light cells were pre- 2. Pevzner, R.A., Comparative Histochemical Study dominantly revealed in certain areas of mesoglea— of Taste Bulbs of Vertebrates, Cand. Sci. Disserta- near neuropil-like clusters and in the vicinity of tion, Leningrad, 1963. developing chemoreceptors. Electron microscop- 3. Aronova, M.Z., Sensory Systems of Comb-Bear- ic photographs indicated their capability for mi- ers, Doctorate Sci. Dissertation, Leningrad, 1987. gration, proliferation, and phagocytosis. These 4. Aronova, M.Z. and Alekseeva, T.M., On Stages of cells have a peculiar shape of their bodies and nu- Evolution of the Locomotor-Sensory System of clei, ultrastructure of the cytoplasm, and the pres- Comb-Bearers, Zh. Evol. Biokhim. Fiziol., 1993, ence of outgrowths and thin processes. We have vol. 29, pp. 663–666. 5. Fedotov, D.M., Evolyutsiya i filogeniya bespoz- succeeded in revealing participation of light cells vonochnykh zhivotnykh (Evolution and Phylogeny in transport into epithelium of the material need- of Invertebrate Animals), Moscow, 1996. ed for replacement of damaged chemoreceptors— 6. Vinogradova, O.S., Neuroscience of the End of the the developing chemoreceptor cells and nerve end- Second Millenium: A Change of Paradigms, Zh. ings. There was shown an important property of Vyssh. Nervn. Deyat. RAN, 2000, vol. 50, pp. 743– the light cells to form synapses with nerve endings 774. and to interact with chemoreceptor axons and, 7. Takenaka, T., Kawakami, T., and Hori, H., Axo- thereby, to presumably affect their growth and plasmic Transport and Its Signal Transduction Me- function. chanism, Jap. J. Physiol., 1998, vol. 48, pp. 413– 420. Thus, the combination of morphological evi- 8. Batchelor, P.E., Liberatore, G.T., and Wong, J.Y., dences allows identifying the light cells as glial cells Activated Macrophages and Microglia Induce that are already present in the nervous system of Dopaminergic Sprouting in the Injured Striatum Ctenophora. Now we know how one of the first and Express Brain-Derived Neurotrophic Factor glial elements looks in representatives of the first and Glia-Derived Neurotrophic Factor, J. Neuro- multicellular animals. sci., 1999, vol. 19, pp. 1708–1716. 9. Barres, B.A., A New Role for Glia: Generation of ACKNOWLEDGMENTS Neurons!, Cell, 1999, vol. 97, pp. 667–670. 10. Lane, A., Structure of Components of the Nervous The authors are grateful to the administration System, Adv. Ins. Physiol., 1981, pp. 1–47. 11. Bobkova, M.V., Structural-Functional Organiza- of the Belomorskaya Biological Station of the Zoo- tion of the Optic System Peripheral Part of the logical Institute of the Russian Academy of Sci- Common Snail Lymnaea stagnalis (L.), Cand. Sci. ences for the possibility to collect experimental Dissertation, St. Petersburg, 1996. material and to Drs. L.V. Zueva, D.V. Lychakov, 12. Levitan, I.B. and Kaczmarek, L.K., The Neuron. G.A. Pyatkina, and N.L. Tumanova for consulta- Cell and Molecular Biology, 3rd Ed., Oxford: Uni- tions and helpful discussion of the obtained results. versity, 2002.

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 40 No. 6 2004