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Acetylcholine-like immunoreactivity in the cestode Hymenolepis diminuta
Brain Research, 513 (1990) 161-165
161
Elsevier BRES 24024
Acetylcholine-like immunoreactivity in the cestode Hymenolepis diminuta S. Ida Samii and Rodney A. Webb Department of Biology, York University, North York, Ont. (Canada)
(Accepted 12 December 1989) Key words: Acetylcholine;Immunoreactivity;Hymenolepis diminuta; Nervous system; Neurotransmitter
The localization of acetylcholinein tissues of the cestode Hymenolepis diminuta was determined, followingderivatization, using an antibody raised against choline-glutaraldehyde-protein.Specificimmunoreactivitywas observed in the rostellum and beneath the suckers of the scolex, in the longitudinal nerve cords, in cells adjacent to some deep longitudinal muscles, in the genital primordium, in the wall of the cirrus sac, and in the external and internal seminal vesicle. The distribution of acetylcholine-likeimmunoreactivityin relation to that of serotonin and glutamate, and the distribution of acetylcholinesterasein H. diminuta is discussed. Acetylcholine (ACh) is found in a variety of organisms, including bacteria, fungi, plants, and animals 1~. Although the functional significance of ACh as a neurotransmitter in vertebrates has been established, there is less direct evidence for the role of ACh in most invertebrates. In parasitic helminths, several lines of evidence suggest that ACh may act as an inhibitory neurotransmitter. For example, ACh and its synthetic and degradative enzymes choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) have been biochemically identified 2'3'6 and AChE has been histochemically localized 17,24 in several platyhelminths. Moreover, levels of choline, ACh, and activities of ChAT and AChE have been measured in homogenates of the rat tapeworm Hymenolepis diminuta by radioenzymatic techniques is. In addition, pharmacological studies using ACh, ACh agonists and antagonists, as well as enzyme inhibitors, suggest that ACh and cholinomimetic drugs exert an inhibitory effect on muscle activity3'16 through receptors which display mixed nicotinic and muscarinic properties TM. However, no attempt has been made to directly localize ACh in the nervous tissue of these organisms. The present study is the first to utilize an immunohistochemical technique using an antibody 4,5 to localize sites of ACh-like immunoreactivity in H. diminuta. The objectives were to identify the localization, morphology and ontogeny of ACh-like immunoreactivity, and to determine if ACh distribution anatomically corresponds to the nervous system. Mature specimens of H. diminuta were recovered from the rat intestine, and the worm tissues were fixed
according to the technique of McRae-Degueurce and Geffard 12. Briefly, tissue sections were washed in 0.1 M cacodylate buffer containing 1% w/v sodium metabisulfite (SMB) (pH 6.2) and subsequently fixed for 1 h in equal volumes of solution A, which consisted of 0.1 M cacodylate and 1 M allyl alcohol (pH 12.4), and solution B, which consisted of 0.1 M cacodylate, SMB (1% w/v) and glutaraldehyde (4% w/v) (pH 7.5). Sequentially, the choline moiety of ACh is released and cross-linked to form a conjugate 4. Fixation was continued for 30 min in solution B, followed by four 10-min washes in Tris-SMB (Tris 0.05 M, SMB 8.5% w/v, pH 7.5). Tissues were subsequently dehydrated and embedded in Paraplast. For immunohistochemistry, 8-/tm sections were cut and mounted on chromalum-coated slides, dewaxed, and rehydrated. Non-specific binding was eliminated by incubation with 3% non-immune goat serum (Dayrnar Laboratories). Following several washes in Tris-SMB buffer, the tissue sections were incubated with a 1:200 dilution of the primary antibody (Biosoft, France) for 24 h. The primary antibody was a rabbit antiserum raised against a choline-glutaraldehyde-protein conjugate 4,5. The sections were washed in Tris-NaCl buffer (Tris 0.05 M, NaC1 8.5% w/v, pH 7.5) and subsequently incubated with a 1:50 dilution of goat anti-rabbit IgG serum (Daymar Laboratories) in Tris-NaCl buffer. Following the peroxidase-antiperoxidase (PAP) technique 15, the reaction product was intensified with osmium tetroxide. The sections were washed, dehydrated mounted in Permount, coverslipped, and examined with a Zeiss photomicroscope. The specificity of the antibody has been tested by
Correspondence: R.A. Webb, Department of Biology, York University, 4700 Keele Street, North York, Ontario M3J 1P3, Canada.
162 ELISA 4 and the highest cross-reactivity was found for choline-glutaraldehyde-protein and acetylcholine-allyl alcohol-glutaraidehyde-protein (choline generated from ACh by allyl alcohol, cross-linked to protein by glutaraldehyde) conjugates, respectively. Controls for the worm tissues were made by omission of the primary or secondary antisera, or the PAP complex, all of which resulted in the elimination of staining. Preadsorption of the primary antiserum with ACh, fixed by the allyl alcohol-glutaraldehyde fixative procedure on proteincovered slides, resulted in almost complete loss of staining of tissues of H. d i m i n u t a . This suggested that the sites of immunoreactivity resulted from an ester of choline and were not simply free choline. Comparable results were found for the antiserum preincubated with succinylcholine, indicating that the choline moiety from a choline ester can potentially be fixed and recognized in the tissue by the antibody. Therefore, we refer to the immunoreactivity as ACh-like immunoreactivity to account for the uncertainty regarding the origin of choline ester, which is presumed to be ACh. The ACh immunohistochemistry for H. d i m i n u t a was performed on all regions of the organism. The scolex (head, or holdfast) is the major anchorage point to the host intestine, and is considered as the center of sensory and motor coordinative activities m. It consists of an apical reduced rostellum, 4 suckers, and contains the CNS of the organism. ACh-like immunoreactivity was concentrated around the suckers and the rostellar capsule. Uni- and bi- or multipolar cells located at the base or around each sucker (2-4 /tin diameter), sent fine processes to the musculature of the suckers (Fig. 1). Two prominent immunoreactive areas, presumably the rostellar ganglia 24, were observed in the rostellum almost equidistant from the immunoreactive 'nerve ring'-like structures around the rostellum. The cerebral ganglia contained very few immunoreactive cells suggesting little involvement of ACh in central (cerebral ganglionic) integration. Extending posteriorly from the cerebral ganglia are the two lateral longitudinal nerve cords which pass down the strobila and are connected by transverse commissures. The longitudinal nerve cords exhibited increasing immunoreactivity from the neck through the immature to the mature progiottids. However, ACh-like immunoreactivity was not observed in the commissures in the strobila. Unlike the weak and diffuse staining observed in the neck region, the immature proglottids displayed immunoreactive cell bodies (3-4/~m in diameter), varicosities (1-2/~m in diameter) and processes (<4 ~m in diameter) within the longitudinal nerve cords. The uni- and multipolar cell bodies were in general, loosely organized
around the outer region of the core of neurites (Fig. 2) in a pattern similar to the ultrastructural organization of nerve cells previously observed in the neck region of H. m i c r o s t o m a 21 .
The highest degree of immunoreactivity, in terms of intensity and distribution, was found in the mature proglottids in which immunoreactive cells and processes were found outside as well as inside the nerve cords. The longitudinal nerve cords contained aggregations of intensely immunoreactive cell bodies (3-6/,m in diameter), of various polarities, with processes seldom extending beyond the nerve cords. The immunoreactive cells outside the longitudinal nerve cords, were larger (4-8 ktm in diameter), and appeared as scattered single cells or, more frequently, as complexes of two or more cells in the dorsolateral and ventrolateral regions. Cell processes were seen to pass close to, or surround the deep longitudinal muscle blocks (Fig. 3). Processes from these cells could sometimes be traced to the longitudinal nerve cords (Fig. 4). The absence of ACh-like immunoreactivity at the level of the superficial circular and longitudinal muscles, and the occurrence of numerous ACh-like immunoreactive processes in the nerve cords may point to a sarconeural model of innervation. In this mode of innervation, which has been previously observed in H. m i c r o s t o m a 19, rather than the nerves growing to the muscles, the sarcoplasmic processes grow and extend to the nerve cords. Furthermore, lack of glial tissue and a distinct perineurium 21 may, in part, explain the diffuse nature of the ACh-like immunoreactivity in the neurons in and around the nerve cord. The ACh-like immunoreactivity was also found in association with the reproductive structures. In the germinative neck region, the centrally placed reproductive primordia appeared as masses of moderately stained cells. In mature proglottids, sections through the wall of the cirrus sac revealed immunoreactive fibers lying on the musculature of the wall, some of which terminated in bulbous varicosities. Occasionally these fibers were traced to a single cell at the juncture of the external and internal seminal vesicles (Fig. 5). Spermatozoa in the internal and external seminal vesicles were observed to be immunoreactive in tissues fixed in glutaraldehyde alone, suggesting that they contain a high content of choline. The function of ACh-like substances in the reproductive primordia, cirrus sac and internal seminal vesicle is not known, but ACh is possibly involved as an excitatory agent in contraction of the internal seminal vesicle, or in protrusion of the cirrus. Such action would be in agreement with the dual action of ACh seen in the vertebrates. On the other hand, ACh, or high concentrations of choline, may be involved in the differentiation
163 nergic nervous tissue in the e m b r y o n i c oncospheres. C o m p a r i s o n of the locales of A C h - l i k e i m m u n o r e a c tivity with those of the enzyme A C h E 24 p r e s e n t e d both similarities and differences. H o w e v e r , because A C h E is not exclusively associated with the nervous system or cholinergic neurons 1'7, distribution of A C h E cannot be considered as either a definitive distribution of neural elements, or as a definitive m a r k e r for A C h . Similarly,
of the p r i m o r d i a l cells to specialized reproductive structures. A C h - l i k e intense staining was o b s e r v e d in 2 - 4 loci of the shelled hexacanth e m b r y o s occupying the uterus of the gravid proglottids (Fig. 6). This result parallels the detection of A C h E activity in H. diminuta embryos TM. These cells were not stained in control incubations, and m a y t h e r e f o r e r e p r e s e n t sites of developing acetylcholi-
i~~¸
i
4 Figs. 1-4. Fig. 1. Oblique section through 2 of the 4 suckers of H. diminuta showing unipolar (curved arrow) and multipolar (arrow) ACh-like immunoreactive cells beneath the suckers (S). Fig. 2. A transverse section through the immature region of the strobila showing a unipolar ACh-like immunoreactive neuron (arrow) in the longitudinal nerve cord (LNC). Fig. 3. A section through the mature region of the strobila displaying the intensely ACh-like immunoreactive longitudinal nerve cord (LNC) and some ACh-like immunoreactive cells (small arrows) adjacent to some muscle blocks of the deep longitudinal muscles (M). The longitudinal nerve cords were only weakly immunoreactive when immune serum, preadsorbed by prior incubation with ACh fixed to protein by allyl alcohol and glutaraldehyde, was used as the first antibody. Under these conditions, the small cells adjacent to the muscle blocks were not found. The tissues do not display any reaction product in the absence of the first antibody, showing that endogenous peroxidase is absent. Fig. 4. A camera lucida drawing of an ACh-like immunoreactive cell, adjacent to a deep longitudinal muscle block (M), with a projection coursing to the longitudinal nerve cord (LNC).
164
Figs. 5,6. Fig. 5. A section through the cirrus in a mature segment of the strobila showing immunoreactive spermatozoa in the external seminal vesicle (ESV). The spermatozoa were also immunoreactive in tissues fixed in glutaraldehyde only, indicating that they may contain a high content of choline. The ACh-like immunoreactive cell body (C) and projecting fibers (small arrows) seen in the wall of the cirrus sac (CS), were not present in tissues fixed in only glutaraldehyde. Fig. 6. ACh-like immunofeactivity (arrows) observed in the oncosphere embryos in the eggs from the uterus of gravid segments of tt. diminuta. although Wilson and Schiller 24 described A C h E activity in the cerebral ganglia of H. diminuta, in the present study, the cerebral ganglia did not exhibit high levels of A C h - l i k e immunoreactivity. F u r t h e r m o r e , while 'bipolar nerves '24 were not detected by the immunohistochemical technique at the level of the germinative neck region, masses of A C h - l i k e immunoreactive cells corresponding to the reproductive p r i m o r d i a were observed. Although sites described by Wilson and Schiller24 as ' m o t o r end plates' were not o b s e r v e d in the present study, such sites may c o r r e s p o n d to areas of concentrated sarconeural innervation in or adjacent to the longitudinal nerve cords 19. B a s e d on the similar pattern of localization of A C h E and serotonin cell bodies, commissures, and collaterals, some investigators 9"13'23 have suggested that serotonin and A C h may act as antagonistic neuromuscular transmitters in H. diminuta and other platyhelminths. The A C h - l i k e immunoreactivity and serotonin-like immuno1 Bradford, H.F., Chemical Neurobiology, Freeman, New York, 1986, pp. 171-172. 2 Bueding, E., Acetylcholinesterase activity of Schistosoma mansoni, Br. J. Pharmacol., 7 (1952) 563-567. 3 Chance, M.R.A. and Mansour, T.E., A contribution to the pharmacology of movement in the liver fluke, Br. J. Pharmacol., 8 (1953) 134-138. 4 Geffard, M., McRae-Degueurce, A. and Souan, M.L., Immunocytochemical detection of acetylcholine in the rat central nervous system, Science, 229 (1985) 77-79. 5 Geffard, M., Vieillemarige, J., Heinrich-Rock, A. and Duris, P., Anti-acetylcholine antibodies and first immunocytochemical application in insect brain, Neurosci. Lett., 57 (1985) 1-6.
reactivity 23, however, displayed differences both in appearance and location. For e x a m p l e , the serotonincontaining commissures and en passant varicosities seen at the level of the superficial longitudinal muscles and the p a r e n c h y m a 23, did not display A C h - l i k e immunoreactivity. On the other hand, glutamate, the most recent excitatory neurotransmitter candidate in platylhelminths 8~2°'22, is, in part, distributed in a manner similar to the A C h - l i k e immunoreactivity seen in the present study. F u r t h e r m o r e , because both glutamate and A C h - l i k e immunoreactivity are found in abundance in and around the longitudinal nerve cords, and these are sites of polyneuronal sarconeural innervation 19, neurally released A C h may antagonize glutamate-stimulated excitatory muscle activity.
This work was supported by Grant A6508 from the Natural Science and Engineering Research Council of Canada to R.A.W. 6 Graff, D.J. and Read, C.P., Specific acetylcholinesterase in Hymenolepis diminuta, J. Parasitol., 53 (1967) 1030-1031. 7 K~isa, P., The cholinergic systems in brain and spinal cord, Prog. Neurobiol., 26 (1986) 2ll-272. 8 Keenan, L. and Koopowitz, H., Physiology and in situ identification of putative aminergic neurotransmitters in the nervous system of Gyrocyte fimbriata, a parasitic flatworm, J. Neurobiol.. 13 (1982) 9-12. 9 Lee, M.B., Bueding, E. and Schiller, E.L., The occurrence and distribution of 5-hydroxytryptamine in Hymenolepis diminuta and H. nana, J. Parasitol, 64 (1978) 257-264. 10 Lumsden, R.D. and Specian, R., The morphology, histology, and fine structure of the adult stage of the cyclophyllidean
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11 12
13
14 15
16
tapeworm Hymenolepis diminuta. In H.P. Arai (Ed.), Biology of the Tapeworm Hymenolepis diminuta, Academic, New York, 1980, pp. 157-280. Macintosh, EC., Acetylcholine. In G.J. Siegel, R.W. AIbers, B.W. Agranoff and R. Katzman (Eds.), Basic Neurochemistry, 3rd edn., Little, Brown, Boston, 1981, pp. 183-204. McRae-Degueurce, A. and Geffard, M., One perfusion mixture for immunocytochemical detection of noradrenalin, dopamine, serotonin, and acetylcholine in the same rat brain, Brain Research, 376 (1986) 217-219. Mettrick, D.E, Interactions between parasites and their hosts: metabolic aspects. In M.J. Howell (Ed.), Proceedings of the 6th International Congress of Parasitology, Pergamon, New York, 1987, pp. 111-117. Rybicka, K., Embryogenesis in Hymenolepis diminuta V. Acetylcholinesterase in embryo, Exp. Parasitol., 20 (1967) 263-266. Sternberger, L.A., Hardy, Jr., EH., Cuculis J.J. and Meyer, H.G., The unlabeled antibody-enzyme method of immunohistochemistry. Preparation and properties of soluble antigenantibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in the identification of spirochetes, J. Histochem. Cytochem., 18 (1970) 315-333. Sukhdeo, M.V.K., Hsu, S.C., Thompson, C.S. and Mettrick, D.E, Hymenolepis diminuta behavioral effects of 5-hydroxytryptamine, acetylcholine, histamine, and somatostatin, J. ParasitoL, 70 (1984) 682-688.
17 Sukhdeo, S.C., Sukhdeo, M.V.K. and Mettrick, D.F., Histochemical localization of acetylcholinesterase in the cerebral ganglion of Fasciola hepatica, a parasitic flatworm, J. ParasitoL, 74 (1988) 1023-1032. 18 Thompson, C.S., Sangster, N.C. and Mettrick, D.F., Cholinergic inhibition of muscle contraction in Hymenolepis diminuta (Cestoda), Can. J. Zool., 64 (1986) 2111-2115. 19 Webb, R.A., Innervation of muscle in the cestode Hymenolepis microstoma, Can. J. Zool., 65 (1987) 928-935. 20 Webb, R.A., Release of exogenously supplied [3H]glutamate and endogenous glutamate from tissue slices of the cestode Hymenolepis diminuta, Can. J. Physiol. Pharmacol., 66 (1988) 889-894. 21 Webb, R.A. and Davey, K.G., The fine structure of the nervous tissue of the metacestode of Hymenolepis microstoma, Can. J. Zool., 54 (1976) 1206-1222. 22 Webb, R.A. and Eklove, H., Demonstration of intense glutamate-like immunoreactivity in the longitudinal nerve cords of the cestode Hymenolepis diminuta, Parasitol Res., 75 (1989) 545548. 23 Webb, R.A. and Mizukawa, K., Serotonin-like immunoreactivity in the cestode Hymenolepis diminuta, J. Comp. Neurol., 234 (1985) 431-440. 24 Wilson, V.C.L.C. and Schiller, E.L., The neuroanatomy of Hymenolepis diminuta and H. nana, J. Parasitol., 55 (1969) 261-270.
161
Elsevier BRES 24024
Acetylcholine-like immunoreactivity in the cestode Hymenolepis diminuta S. Ida Samii and Rodney A. Webb Department of Biology, York University, North York, Ont. (Canada)
(Accepted 12 December 1989) Key words: Acetylcholine;Immunoreactivity;Hymenolepis diminuta; Nervous system; Neurotransmitter
The localization of acetylcholinein tissues of the cestode Hymenolepis diminuta was determined, followingderivatization, using an antibody raised against choline-glutaraldehyde-protein.Specificimmunoreactivitywas observed in the rostellum and beneath the suckers of the scolex, in the longitudinal nerve cords, in cells adjacent to some deep longitudinal muscles, in the genital primordium, in the wall of the cirrus sac, and in the external and internal seminal vesicle. The distribution of acetylcholine-likeimmunoreactivityin relation to that of serotonin and glutamate, and the distribution of acetylcholinesterasein H. diminuta is discussed. Acetylcholine (ACh) is found in a variety of organisms, including bacteria, fungi, plants, and animals 1~. Although the functional significance of ACh as a neurotransmitter in vertebrates has been established, there is less direct evidence for the role of ACh in most invertebrates. In parasitic helminths, several lines of evidence suggest that ACh may act as an inhibitory neurotransmitter. For example, ACh and its synthetic and degradative enzymes choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) have been biochemically identified 2'3'6 and AChE has been histochemically localized 17,24 in several platyhelminths. Moreover, levels of choline, ACh, and activities of ChAT and AChE have been measured in homogenates of the rat tapeworm Hymenolepis diminuta by radioenzymatic techniques is. In addition, pharmacological studies using ACh, ACh agonists and antagonists, as well as enzyme inhibitors, suggest that ACh and cholinomimetic drugs exert an inhibitory effect on muscle activity3'16 through receptors which display mixed nicotinic and muscarinic properties TM. However, no attempt has been made to directly localize ACh in the nervous tissue of these organisms. The present study is the first to utilize an immunohistochemical technique using an antibody 4,5 to localize sites of ACh-like immunoreactivity in H. diminuta. The objectives were to identify the localization, morphology and ontogeny of ACh-like immunoreactivity, and to determine if ACh distribution anatomically corresponds to the nervous system. Mature specimens of H. diminuta were recovered from the rat intestine, and the worm tissues were fixed
according to the technique of McRae-Degueurce and Geffard 12. Briefly, tissue sections were washed in 0.1 M cacodylate buffer containing 1% w/v sodium metabisulfite (SMB) (pH 6.2) and subsequently fixed for 1 h in equal volumes of solution A, which consisted of 0.1 M cacodylate and 1 M allyl alcohol (pH 12.4), and solution B, which consisted of 0.1 M cacodylate, SMB (1% w/v) and glutaraldehyde (4% w/v) (pH 7.5). Sequentially, the choline moiety of ACh is released and cross-linked to form a conjugate 4. Fixation was continued for 30 min in solution B, followed by four 10-min washes in Tris-SMB (Tris 0.05 M, SMB 8.5% w/v, pH 7.5). Tissues were subsequently dehydrated and embedded in Paraplast. For immunohistochemistry, 8-/tm sections were cut and mounted on chromalum-coated slides, dewaxed, and rehydrated. Non-specific binding was eliminated by incubation with 3% non-immune goat serum (Dayrnar Laboratories). Following several washes in Tris-SMB buffer, the tissue sections were incubated with a 1:200 dilution of the primary antibody (Biosoft, France) for 24 h. The primary antibody was a rabbit antiserum raised against a choline-glutaraldehyde-protein conjugate 4,5. The sections were washed in Tris-NaCl buffer (Tris 0.05 M, NaC1 8.5% w/v, pH 7.5) and subsequently incubated with a 1:50 dilution of goat anti-rabbit IgG serum (Daymar Laboratories) in Tris-NaCl buffer. Following the peroxidase-antiperoxidase (PAP) technique 15, the reaction product was intensified with osmium tetroxide. The sections were washed, dehydrated mounted in Permount, coverslipped, and examined with a Zeiss photomicroscope. The specificity of the antibody has been tested by
Correspondence: R.A. Webb, Department of Biology, York University, 4700 Keele Street, North York, Ontario M3J 1P3, Canada.
162 ELISA 4 and the highest cross-reactivity was found for choline-glutaraldehyde-protein and acetylcholine-allyl alcohol-glutaraidehyde-protein (choline generated from ACh by allyl alcohol, cross-linked to protein by glutaraldehyde) conjugates, respectively. Controls for the worm tissues were made by omission of the primary or secondary antisera, or the PAP complex, all of which resulted in the elimination of staining. Preadsorption of the primary antiserum with ACh, fixed by the allyl alcohol-glutaraldehyde fixative procedure on proteincovered slides, resulted in almost complete loss of staining of tissues of H. d i m i n u t a . This suggested that the sites of immunoreactivity resulted from an ester of choline and were not simply free choline. Comparable results were found for the antiserum preincubated with succinylcholine, indicating that the choline moiety from a choline ester can potentially be fixed and recognized in the tissue by the antibody. Therefore, we refer to the immunoreactivity as ACh-like immunoreactivity to account for the uncertainty regarding the origin of choline ester, which is presumed to be ACh. The ACh immunohistochemistry for H. d i m i n u t a was performed on all regions of the organism. The scolex (head, or holdfast) is the major anchorage point to the host intestine, and is considered as the center of sensory and motor coordinative activities m. It consists of an apical reduced rostellum, 4 suckers, and contains the CNS of the organism. ACh-like immunoreactivity was concentrated around the suckers and the rostellar capsule. Uni- and bi- or multipolar cells located at the base or around each sucker (2-4 /tin diameter), sent fine processes to the musculature of the suckers (Fig. 1). Two prominent immunoreactive areas, presumably the rostellar ganglia 24, were observed in the rostellum almost equidistant from the immunoreactive 'nerve ring'-like structures around the rostellum. The cerebral ganglia contained very few immunoreactive cells suggesting little involvement of ACh in central (cerebral ganglionic) integration. Extending posteriorly from the cerebral ganglia are the two lateral longitudinal nerve cords which pass down the strobila and are connected by transverse commissures. The longitudinal nerve cords exhibited increasing immunoreactivity from the neck through the immature to the mature progiottids. However, ACh-like immunoreactivity was not observed in the commissures in the strobila. Unlike the weak and diffuse staining observed in the neck region, the immature proglottids displayed immunoreactive cell bodies (3-4/~m in diameter), varicosities (1-2/~m in diameter) and processes (<4 ~m in diameter) within the longitudinal nerve cords. The uni- and multipolar cell bodies were in general, loosely organized
around the outer region of the core of neurites (Fig. 2) in a pattern similar to the ultrastructural organization of nerve cells previously observed in the neck region of H. m i c r o s t o m a 21 .
The highest degree of immunoreactivity, in terms of intensity and distribution, was found in the mature proglottids in which immunoreactive cells and processes were found outside as well as inside the nerve cords. The longitudinal nerve cords contained aggregations of intensely immunoreactive cell bodies (3-6/,m in diameter), of various polarities, with processes seldom extending beyond the nerve cords. The immunoreactive cells outside the longitudinal nerve cords, were larger (4-8 ktm in diameter), and appeared as scattered single cells or, more frequently, as complexes of two or more cells in the dorsolateral and ventrolateral regions. Cell processes were seen to pass close to, or surround the deep longitudinal muscle blocks (Fig. 3). Processes from these cells could sometimes be traced to the longitudinal nerve cords (Fig. 4). The absence of ACh-like immunoreactivity at the level of the superficial circular and longitudinal muscles, and the occurrence of numerous ACh-like immunoreactive processes in the nerve cords may point to a sarconeural model of innervation. In this mode of innervation, which has been previously observed in H. m i c r o s t o m a 19, rather than the nerves growing to the muscles, the sarcoplasmic processes grow and extend to the nerve cords. Furthermore, lack of glial tissue and a distinct perineurium 21 may, in part, explain the diffuse nature of the ACh-like immunoreactivity in the neurons in and around the nerve cord. The ACh-like immunoreactivity was also found in association with the reproductive structures. In the germinative neck region, the centrally placed reproductive primordia appeared as masses of moderately stained cells. In mature proglottids, sections through the wall of the cirrus sac revealed immunoreactive fibers lying on the musculature of the wall, some of which terminated in bulbous varicosities. Occasionally these fibers were traced to a single cell at the juncture of the external and internal seminal vesicles (Fig. 5). Spermatozoa in the internal and external seminal vesicles were observed to be immunoreactive in tissues fixed in glutaraldehyde alone, suggesting that they contain a high content of choline. The function of ACh-like substances in the reproductive primordia, cirrus sac and internal seminal vesicle is not known, but ACh is possibly involved as an excitatory agent in contraction of the internal seminal vesicle, or in protrusion of the cirrus. Such action would be in agreement with the dual action of ACh seen in the vertebrates. On the other hand, ACh, or high concentrations of choline, may be involved in the differentiation
163 nergic nervous tissue in the e m b r y o n i c oncospheres. C o m p a r i s o n of the locales of A C h - l i k e i m m u n o r e a c tivity with those of the enzyme A C h E 24 p r e s e n t e d both similarities and differences. H o w e v e r , because A C h E is not exclusively associated with the nervous system or cholinergic neurons 1'7, distribution of A C h E cannot be considered as either a definitive distribution of neural elements, or as a definitive m a r k e r for A C h . Similarly,
of the p r i m o r d i a l cells to specialized reproductive structures. A C h - l i k e intense staining was o b s e r v e d in 2 - 4 loci of the shelled hexacanth e m b r y o s occupying the uterus of the gravid proglottids (Fig. 6). This result parallels the detection of A C h E activity in H. diminuta embryos TM. These cells were not stained in control incubations, and m a y t h e r e f o r e r e p r e s e n t sites of developing acetylcholi-
i~~¸
i
4 Figs. 1-4. Fig. 1. Oblique section through 2 of the 4 suckers of H. diminuta showing unipolar (curved arrow) and multipolar (arrow) ACh-like immunoreactive cells beneath the suckers (S). Fig. 2. A transverse section through the immature region of the strobila showing a unipolar ACh-like immunoreactive neuron (arrow) in the longitudinal nerve cord (LNC). Fig. 3. A section through the mature region of the strobila displaying the intensely ACh-like immunoreactive longitudinal nerve cord (LNC) and some ACh-like immunoreactive cells (small arrows) adjacent to some muscle blocks of the deep longitudinal muscles (M). The longitudinal nerve cords were only weakly immunoreactive when immune serum, preadsorbed by prior incubation with ACh fixed to protein by allyl alcohol and glutaraldehyde, was used as the first antibody. Under these conditions, the small cells adjacent to the muscle blocks were not found. The tissues do not display any reaction product in the absence of the first antibody, showing that endogenous peroxidase is absent. Fig. 4. A camera lucida drawing of an ACh-like immunoreactive cell, adjacent to a deep longitudinal muscle block (M), with a projection coursing to the longitudinal nerve cord (LNC).
164
Figs. 5,6. Fig. 5. A section through the cirrus in a mature segment of the strobila showing immunoreactive spermatozoa in the external seminal vesicle (ESV). The spermatozoa were also immunoreactive in tissues fixed in glutaraldehyde only, indicating that they may contain a high content of choline. The ACh-like immunoreactive cell body (C) and projecting fibers (small arrows) seen in the wall of the cirrus sac (CS), were not present in tissues fixed in only glutaraldehyde. Fig. 6. ACh-like immunofeactivity (arrows) observed in the oncosphere embryos in the eggs from the uterus of gravid segments of tt. diminuta. although Wilson and Schiller 24 described A C h E activity in the cerebral ganglia of H. diminuta, in the present study, the cerebral ganglia did not exhibit high levels of A C h - l i k e immunoreactivity. F u r t h e r m o r e , while 'bipolar nerves '24 were not detected by the immunohistochemical technique at the level of the germinative neck region, masses of A C h - l i k e immunoreactive cells corresponding to the reproductive p r i m o r d i a were observed. Although sites described by Wilson and Schiller24 as ' m o t o r end plates' were not o b s e r v e d in the present study, such sites may c o r r e s p o n d to areas of concentrated sarconeural innervation in or adjacent to the longitudinal nerve cords 19. B a s e d on the similar pattern of localization of A C h E and serotonin cell bodies, commissures, and collaterals, some investigators 9"13'23 have suggested that serotonin and A C h may act as antagonistic neuromuscular transmitters in H. diminuta and other platyhelminths. The A C h - l i k e immunoreactivity and serotonin-like immuno1 Bradford, H.F., Chemical Neurobiology, Freeman, New York, 1986, pp. 171-172. 2 Bueding, E., Acetylcholinesterase activity of Schistosoma mansoni, Br. J. Pharmacol., 7 (1952) 563-567. 3 Chance, M.R.A. and Mansour, T.E., A contribution to the pharmacology of movement in the liver fluke, Br. J. Pharmacol., 8 (1953) 134-138. 4 Geffard, M., McRae-Degueurce, A. and Souan, M.L., Immunocytochemical detection of acetylcholine in the rat central nervous system, Science, 229 (1985) 77-79. 5 Geffard, M., Vieillemarige, J., Heinrich-Rock, A. and Duris, P., Anti-acetylcholine antibodies and first immunocytochemical application in insect brain, Neurosci. Lett., 57 (1985) 1-6.
reactivity 23, however, displayed differences both in appearance and location. For e x a m p l e , the serotonincontaining commissures and en passant varicosities seen at the level of the superficial longitudinal muscles and the p a r e n c h y m a 23, did not display A C h - l i k e immunoreactivity. On the other hand, glutamate, the most recent excitatory neurotransmitter candidate in platylhelminths 8~2°'22, is, in part, distributed in a manner similar to the A C h - l i k e immunoreactivity seen in the present study. F u r t h e r m o r e , because both glutamate and A C h - l i k e immunoreactivity are found in abundance in and around the longitudinal nerve cords, and these are sites of polyneuronal sarconeural innervation 19, neurally released A C h may antagonize glutamate-stimulated excitatory muscle activity.
This work was supported by Grant A6508 from the Natural Science and Engineering Research Council of Canada to R.A.W. 6 Graff, D.J. and Read, C.P., Specific acetylcholinesterase in Hymenolepis diminuta, J. Parasitol., 53 (1967) 1030-1031. 7 K~isa, P., The cholinergic systems in brain and spinal cord, Prog. Neurobiol., 26 (1986) 2ll-272. 8 Keenan, L. and Koopowitz, H., Physiology and in situ identification of putative aminergic neurotransmitters in the nervous system of Gyrocyte fimbriata, a parasitic flatworm, J. Neurobiol.. 13 (1982) 9-12. 9 Lee, M.B., Bueding, E. and Schiller, E.L., The occurrence and distribution of 5-hydroxytryptamine in Hymenolepis diminuta and H. nana, J. Parasitol, 64 (1978) 257-264. 10 Lumsden, R.D. and Specian, R., The morphology, histology, and fine structure of the adult stage of the cyclophyllidean
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tapeworm Hymenolepis diminuta. In H.P. Arai (Ed.), Biology of the Tapeworm Hymenolepis diminuta, Academic, New York, 1980, pp. 157-280. Macintosh, EC., Acetylcholine. In G.J. Siegel, R.W. AIbers, B.W. Agranoff and R. Katzman (Eds.), Basic Neurochemistry, 3rd edn., Little, Brown, Boston, 1981, pp. 183-204. McRae-Degueurce, A. and Geffard, M., One perfusion mixture for immunocytochemical detection of noradrenalin, dopamine, serotonin, and acetylcholine in the same rat brain, Brain Research, 376 (1986) 217-219. Mettrick, D.E, Interactions between parasites and their hosts: metabolic aspects. In M.J. Howell (Ed.), Proceedings of the 6th International Congress of Parasitology, Pergamon, New York, 1987, pp. 111-117. Rybicka, K., Embryogenesis in Hymenolepis diminuta V. Acetylcholinesterase in embryo, Exp. Parasitol., 20 (1967) 263-266. Sternberger, L.A., Hardy, Jr., EH., Cuculis J.J. and Meyer, H.G., The unlabeled antibody-enzyme method of immunohistochemistry. Preparation and properties of soluble antigenantibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in the identification of spirochetes, J. Histochem. Cytochem., 18 (1970) 315-333. Sukhdeo, M.V.K., Hsu, S.C., Thompson, C.S. and Mettrick, D.E, Hymenolepis diminuta behavioral effects of 5-hydroxytryptamine, acetylcholine, histamine, and somatostatin, J. ParasitoL, 70 (1984) 682-688.
17 Sukhdeo, S.C., Sukhdeo, M.V.K. and Mettrick, D.F., Histochemical localization of acetylcholinesterase in the cerebral ganglion of Fasciola hepatica, a parasitic flatworm, J. ParasitoL, 74 (1988) 1023-1032. 18 Thompson, C.S., Sangster, N.C. and Mettrick, D.F., Cholinergic inhibition of muscle contraction in Hymenolepis diminuta (Cestoda), Can. J. Zool., 64 (1986) 2111-2115. 19 Webb, R.A., Innervation of muscle in the cestode Hymenolepis microstoma, Can. J. Zool., 65 (1987) 928-935. 20 Webb, R.A., Release of exogenously supplied [3H]glutamate and endogenous glutamate from tissue slices of the cestode Hymenolepis diminuta, Can. J. Physiol. Pharmacol., 66 (1988) 889-894. 21 Webb, R.A. and Davey, K.G., The fine structure of the nervous tissue of the metacestode of Hymenolepis microstoma, Can. J. Zool., 54 (1976) 1206-1222. 22 Webb, R.A. and Eklove, H., Demonstration of intense glutamate-like immunoreactivity in the longitudinal nerve cords of the cestode Hymenolepis diminuta, Parasitol Res., 75 (1989) 545548. 23 Webb, R.A. and Mizukawa, K., Serotonin-like immunoreactivity in the cestode Hymenolepis diminuta, J. Comp. Neurol., 234 (1985) 431-440. 24 Wilson, V.C.L.C. and Schiller, E.L., The neuroanatomy of Hymenolepis diminuta and H. nana, J. Parasitol., 55 (1969) 261-270.