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Immunohistochemical and electrochemical detection of serotonin in the nervous system of Fasciola hepatica, a parasitic flatworm
Brain Research, 463 (1988) 57-62
57
Elsevier BRE 13997
Immunohistochemical and electrochemical detection of serotonin in the nervous system of Fasciola hepatica, a parasitic flatworm Suzanne C. Sukhdeo and Michael V.K. Sukhdeo Department of Zoology, University of Toronto, Toronto, Ont. (Canada) (Accepted 11 May 1988)
Key words: Serotonin; Immunohistochemistry; Cerebral ganglion; Bilateral symmetry
The head region of the trematode parasite Fasciola hepatica contains 3.47 _+0.42 pmol/mg wet wt. of serotonin as measured by highperformance liquid chromatography coupled to electrochemical detection. The head region includes the cerebral ganglia, the transverse commissure and associated nervous tissue that innervates the musculature of the oral sucker, pharynx and body wall. Tissue from the tail, which contains little nervous innervation, has approximately 20 times less serotonin (0.18 + 0.01 pmol/mg wet wt.). Immunohistochemistry was used to identify serotonin-like immunoreactive cells. Bipolar and multipolar cell bodies in the cerebral ganglia show serotonin-like immunoreactivity. Also evident are serotonin-like immunoreactive processes in the neuropile and in the transverse commissure that connects the ganglia, and immunoreactive peripheral bipolar cell bodies innervating the musculature of the pharynx and body wall. The cell bodies containing serotonin are organized in bilateral symmetry with homologous cell bodies and processes represented in each ganglion and on both sides of the pharynx.
INTRODUCTION The Phylum Platyhelminthes (flatworms) contains first seen in the Phylum Platyhelminthes22'29 and the ly symmetrical metazoans and the first animals to possess 'true' brains 5. Brains and bilateral symmetry are features that are retained in subsequent evolutionary development and these organisms occupy a crucial position at the junction of vertebrate and invertebrate phylogenetic radiation I. Serotonin has been recovered from the tissues of several flatworms 3"7"19"22"29 where it is believed to exert biochemical and hormonal effects on carbohydrate metabolism 16'18. A n u m b e r of studies using Falck-Hillarp, radioautographic and immunochemical techniqueg have also localized serotonin in cells of the central and peripheral nervous systems of flatworms 2'8"12"28'3°. There is some evidence that serotonin may function as an excitatory neurotransmitter and although the effects are somewhat inconsistent26, serotonin does increase the motility of several flat-
worm species 15"tT'26`3j. The excitatory effect is believed to be mediated by the peripheral nerves because it is not affected by decerebration 17'26. The functions of the n e u r o n s with serotonin-like i m m u n o reactivity that are found in the cerebral ganglia of flatworms 1°12"13are unclear.
Fasciola hepatica (Trematoda) is a large parasitic flatworm that is a feasible candidate for electrophysiological studies because of its size and the ease of m a i n t e n a n c e in the laboratory. The purpose of this study was: (1) to determine if endogenous serotonin is present in the tissues of F. hepatica using a sensitive and specific assay based on high-performance liquid chromatography; and (2) to map the distribution of serotonin-like neurons in the head region of this organism using an immunohistochemical technique. MATERIALS AND METHODS The infective metacercarial stages of F. hepatica were purchased from a commercial supplier (Bald-
Correspondence: S. Sukhdeo, Department of Zoology, University of Toronto, Toronto, Ont. M5S 1A1, Canada. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
58 win Enterprises, Oregon) and 15-20 metacercariae were given to Wistar rats (Woodlyn Labs., Guelph, Ont.) by intragastric inoculation. Adult worms were collected from the bile ducts of rats infected 4 - 6 months previously and placed in a buffered ringer solution for dissection. The head region, approximately 2 mm in length (containing the oral sucker, pharynx, paired cerebral ganglia, transverse commissure and mesenchyme), was separated from the body and used for immunohistochemistry and HPLC determinations. F, hepatica is an acoelomate and all the organs are embedded in mesenchyme. It is impossible to distinguish between different tissues in vivo and the selective dissection and removal of the cerebral ganglia is technically unfeasible. Two preparations of the head region were made for immunofluorescence experiments, a dorsal and a ventral preparation. In the dorsal preparation, the dorsal tegument was carefully dissected away from the head to expose the underlying tissues to the immunochemical solutions. In the ventral preparation, the ventral tegument was removed.
Electrochemical detection of serotonin Serotonin was assayed by high-performance liquid chromatography (HPLC) using the electrochemical detection procedure of Orchard et al. el. The head regions of the organisms were used for the HPLC assay but the dorsal and ventral tegumental coverings were left intact. Tissue from the tail region, of similar size and weight to the head region, were also examined. The tail region contains little nervous innervation. The protocol for the preparation of the tissue for HPLC was modified from the procedure described by Orchard et al. 21. Briefly, 3 heads or 3 tails were pooled, weighed (approximate weight 8 mg) and placed in 30/A of ice cold 0.2 N perchloric acid and frozen a t - 2 0 °C. The tissue was thawed and 175/~1 of HPLC buffer was added. The tissue was sonicated in the mixture for 30 s, centrifuged at 8800 g for 5 - 7 minutes and then filtered through a 0.22 ktm nylon filter. A 100/~1 aliquot of the filtered supernatant was injected onto a Brownlee RP-18 Spheri-5 HPLC column (4.6 mm x 22 cm). The detection of the eluted compounds was done electrochemically using an ESA model 5100 A detection system coupled to a model 5010 dual coulometric detector. Serotonin was
detected on the second detector set at 0.35 V with the first detector set at 0.1 V acting as a screen. Serotonin was quantified using the external standard method incorporated into an SP4700 integrator. The tissues for each replicate (3 heads or 3 tails per replicate) were spiked with serotonin to confirm the identity of the oxidizable substance and to check for losses.
Serotonin immunohistochemistry The head preparations were fixed for 40 min at room temperature in 2% paraformaldehyde in Millonigs buffer (pH 7.4). After fixation, the preparations were washed for 3 - 4 h in phosphate-buffered saline (PBS) (pH 7.2) which contained 0.9% NaCI. The preparations were then incubated for 1 h in PBS containing 4% Triton X-100, 2% bovine serum albumin and 2% normal goat serum. The anti-serotonin antiserum (lmmuno Nuclear Corp., Stillwater, MN) was diluted 1:1000 in PBS containing 2% bovine serum albumin, 0.4% Triton X-100 and 2% normal goat serum and incubated at 4 °C for 18 h. The preparations were then incubated in the diluted antiserum for 48 h at 4 °C with constant agitation. After antibody incubation, the preparations were washed for 3-4 h with PBS and incubated overnight at 4 °C with fluorescent isothiocyanate-labelled sheep anti-rabbit IgG (Daymar Labs., Toronto, Ont.) diluted 1:200 in 10% normal goat serum in PBS. The preparations were then washed for 2-3 h in PBS and mounted in 5% n-propylgallate made up in glycerol (pH 7.3) in well-slides. The preparations were examined for serotoninlike immunoreactivity with an epi-illumination fluorescent microscope. The data on the distribution of serotonin-like immunoreactivity are based on 6 dorsal and 6 ventral replicates. In addition, 7 control replicates were done to test for the antiserum specificity. The control preparations were incubated with serotonin antiserum that had been preabsorbed with 1 mg/ml serotonin or 100 #g/ml serotonin conjugated to bovine serum albumin (lmmuno Nuclear Corp., Stillwater, MN) for 18 h at 4 °C. RESULTS
Electrochemical detection of serotonin The head region of F. hepatica, which includes the cerebral ganglia and associated nervous tissue, the
59 oral s u c k e r
and
pharynx,
contains
3.47
+
0.42
Cerebral ganglia
p m o l / m g wet wt. of s e r o t o n i n (n = 4). T h e tail re-
N e u r o n s and their processes that w e r e f o u n d to be
gion, which d o e s not c o n t a i n significant n e r v o u s in-
consistently i m m u n o r e a c t i v e are s u m m a r i z e d in c o m -
n e r v a t i o n , has a p p r o x i m a t e l y 20 times less s e r o t o n i n
posite drawings of the dorsal and v e n t r a l p r e p a r a -
(0.18 + 0.01 p m o l / m g wet wt; n = 4).
tions (Fig. 1). All staining was absent in c o n t r o l p r e p -
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Fig. 1. Serotonin-like immunoreactive cells and processes in the head region of Fasciola hepatica. These diagrams represent composites of camera lucida drawings of cells that reacted consistently with the serotonin antiserum. Five cerebral ganglion cell bodies (GCb) were observed from the dorsal (A) view and 3 could be seen in the ventral (B) view. The ganglion cell bodies extend processes into their respective neuropile area (Nf), anterior to innervate the oral sucker (O.S.), posterior to innervate the pharynx (Ph) and contralateral processes through the dorsal transverse commissure (COM, A). Peripheral cell bodies (Cb) innervating the pharynx (Ph) also send contralateral nerve processes over the dorsal (A) and ventral (B) surfaces of the pharynx.
60
Fig. 2. Whole mount preparations showing serotonin-like immunoreactive cells and nerve processes in the head region of F. hepatica. A: dorsal view of the head showing two immunofluorescent areas which represent the paired cerebral ganglia (Curved arrows) on either side of the junction between the oral sucker (OS) and the pharynx (P), (E = egg), scale bar = 20/~m; B: higher magnification of one cerebral ganglion showing two immunoreactive ganglion cell bodies in focus (Cb) and a meshwork of immunoreactive nerve fibers of the neuropile (curved arrows). One process (arrowhead) extends anterior to innervate the oral sucker, scale bar = 10 urn; C: a bipolar ganglion cell with stained nucleus (N) and process (arrowhead) extending towards the oral sucker, scale bar = 10gtm; D: two cell bodies (arrows) situated on either side of the pharynx sending contralateral processes (arrowhead) over the pharynx midline (PM, large arrow), scale bar = 2t)/~m.
61 arations where the serotonin antiserum was pre-absorbed with serotonin-conjugated to BSA. Homologous bilaterally symmetrical neurons showing serotonin-like immunoreactivity occur in both the dorsal and ventral aspects of the cerebral ganglia (Figs. 1 and 2A). Five serotonin neurons occur in each ganglion and 3 of these can be seen from the ventral aspect (Figs. 1 and 2B). The majority of these neurons appear spindle-shaped with longitudinal axes of 25-30 ~m (Figs. 1 and 2B). The neurons are a mixture of bipolar, multipolar and monopolar types with nerve processes that extend out of the ganglia to innervate the musculature of the oral sucker and pharynx (Figs. 1 and 2D). Neurites also extend into the neuropile of the ganglia and the immunoreactive neurons of the ganglia surround a core of finely stained processes in the neuropile (Figs. 1 and 2B). Six to 10 serotonin immunoreactive processes connect the ganglia via the transverse commissure (Fig. 1A).
fore, one may be reasonably confident that the cell bodies and processes described in this study do contain serotonin. The serotonin-like immunoreactive neurons in the cerebral ganglia of F. hepatica are bilaterally symmetrical with 5 neurons occupying similar locations in each ganglion. Some of the nerve processes from these neurons clearly extend to the contralateral ganglion via the transverse commissure. The bilateral symmetry of homologous serotonin immunoreactive neurons that extend contralateral processes is also seen in the peripheral neurons innervating the pharynx. In this study of F. hepatica and in other studies of flatworms, many of the serotonin-containing neurons appear to be motor neurons that are responsible for innervation of the musculature of the digestive tract and body wall 3°. Although not fully substantiated by electrophysiological data, this preferential localization of serotonin in the motor neurons of invertebrates has been reported by several investigatorsll'22
Peripheral immunoreactive cells
We believe that the bilaterally symmetrical distribution of serotonergic neurons may have significant functional implications in the evolution of the serotonin system. Serotonin has been recovered from the tissues of representatives of most animal phyla 6A4, but the association of serotonin with nervous tissue is first seen in the Phylum Platyhelminthes 22'29 and the occurrence of serotonin neurons coincides with the development of bilateral symmetry. The bilaterally symmetrical distribution of ganglionic serotonergic neurons described in this study of F. hepatica is not restricted to this flatworm species. The Falck-Hillarp technique shows bilaterally symmetrical serotonin cells with contralateral processes in the cerebral ganglia of several free-living flatworms (Turbellaria) 3° and a bilateral symmetry of serotonergic structures in the ganglia of parasitic flatworms (Trematoda and Cestoda) 2'15. This pattern of bilateral symmetry and contralateral processes of serotonergic neurons is conserved in the higher invertebrate phyla and is seen, for example, in the giant serotonin-containing cells in ganglia of molluscs 23 and in the serotonergic neurons in the brains and ganglia of many annelid 23"24 and insect species 4,z°. We suggest that the pattern of homologous bilaterally symmetrical ganglionic neurons, that extend contralateral processes, reflects a primitive function of coordination of muscular activi-
There are numerous small bipolar neurons (10-15 ~tm longitudinal axis) (Fig. 2C) showing serotoninlike immunoreactivity that are not associated with the cerebral ganglia. Many of these neurons innervate the pharynx where they are organized in bilateral symmetry and typically send contralateral processes around the pharynx (Figs. 1 and 2D). Similar bilaterally symmetrical, bipolar neurons with their neurites oriented to the longitudinal axis of the worm innervate the musculature of the body wall of the animal. DISCUSSION The serotonin antiserum used in the present study has been used in many immunochemical studies and is reported to have high specificity with little crossreactivitf 5,27. This immunohistochemical staining in itself does not constitute chemical identification of serotonin; however, pre-absorption of the antiserum with serotonin conjugated to bovine serum albumin abolishes all staining. In addition, HPLC coupled to electrochemical detection (an accurate and sensitive assay 9) identifies the presence of serotonin in the tisrues of F. hepatica and suggests that this amine is con:entrated in the head region of this organism. There-
62 ty t h a t e v o l v e d in t h e first b i l a t e r a l l y s y m m e t r i c a l organisms.
a n d A n g e l a L a n g e for p e r f o r m i n g t h e H P L C a n a l y s i s and assistance with the immunohistochemistry. This s t u d y was s u p p o r t e d by a n O n t a r i o G r a d u a t e S c h o l -
ACKNOWLEDGEMENTS
a r s h i p to S.C.S. a n d N S E R C
G r a n t A 4 6 6 7 to D . F .
Mettrick. T h e a u t h o r s w o u l d like to t h a n k D r s . Ian O r c h a r d
REFERENCES 1 Barnes. R.D., Invertebrate Zoology, 5th Edn., Saunders College Publishing, Toronto, 1987, 893 pp.. 2 Bennett. J. and Bueding, E., Localization of biogenic' amines in Schistosoma mansoni, Comp. Biochem. Physiol., 39 (1971) 859-867. 3 Bennett, J., Timms, A.R. and Engstrom, R.G., Occurrence and levels of 5-hydroxytryptamine in Schistosoma mansoni, Mol. Pharmacol., 5 (1969)542-545. 4 Bishop, C.A. and O'Shea, M., Serotonin immunoreactive neurons in the central nervous system of an insect (Periplaneta americana). J. Neurobiol., 14 (1983) 251-269. 5 Bullock, T.H. and Horridge, G.A., Structure and Function in the Nervous Systems of Invertebrates. Vol 1, Freeman, San Francisco, 1965. pp. 535-577. 6 Carlberg, M. and Rosengren, E., Biochemical basis for adrenergie neurotransmission in coelenterates, J. Comp. Physiol. B., 155 (1985) 251-255. 7 Chou, T.-C.T., Bennett, J. and Bueding. E., Occurrence and concentrations of biogenic amines in trematodes, J. Parasitol.. 58 ( 19721 1098-1102. 8 Dei-Cas, E., Dhainaut-Courtois, N., Dhainant, A. and Vernes, A., Ultrastructural localization of tritiated 5-HT in adult Schistosoma mansoni, Biol. Cell., 35 (1979) 321-324. 9 Downer, R.G.H.. Bailey, B.A. and Martin, R.J., Estimation of biogenic amines in biological tissues. In R. Gilles and J. Balthazart (Eds.), Neurobiology, Springer, Berlin, 1985, pp. 248-263. 10 Fairweather, I., Maule, A.G., Mitchell S.H., Johnston, C.F. and Halton, D.W., Immunocytochemical demonstration of 5-hydroxytryptamine (serotonin) in the nervous system of the liver fluke, Fasciola hepatica (Trematoda, Digenea), Parasitol. Res., 73 (1987) 255-258. 11 Gerschenfeld, H.M., Chemical transmission in invertebrate central nervous systems and neuromuscular junctions, Physiol. Rev., 53 ( 19731 1-119. 12 Gustafsson, M.K.S., lmmonocytochemical demonstration of neuropeptides and serotonin in the nervous system of adult Schistosoma mansoni. Parasitol. Res.. 74 (1987) 168-174. 13 Halton, D.W., Maule, A.G., Johnston, C.F. and Fairweather, I., Occurrence of 5-hydroxytryptamine (serotonin) in the nervous system of a monogenean Diclodophora merlangi, Parasitol. Res., 74 (1987) 151-154. 14 Klemm, N., The distribution of biogenic monoamines in invertebrates. In R. Gilles and J. Balthazart (Eds.), Neurobiology, Springer, Berlin, 1985, pp. 280-296. 15 Lee, M.B., Bueding, E. and Schiller, E.L., The occurrence and distribution of 5-hydroxytryptamine in ttymenolepis diminuta and H. nana, J. Parasitol., 64 (19781 257-264. 16 Mansour, J.M. and Mansour, T.E., GTP-binding proteins associated with serotonin-activated adenylate cyclase in Fasciola hepatica. Mol. Biochem. Parasitol., 21 (1986) 139-
149. 17 Mansour, T.E., The effects of lysergic acid diethylamide, 5hydroxytryptamine, and related compounds on the liver fluke, Fasciola hepatica, Br. J. Pharmacol., 12 (1957) 406-409. 18 Mansour, T.E., Serotonin receptors in parasitic worms, Adv. Parasitol., 23 (1984) 1-37. 19 Mansour, T.E. and Stone, D.B., Biochemical effects of lysergic acid diethylamide on the liver fluke Fasciola hepatica, Biochem. Pharmacol. , 19 (1970) 1137-1146. 20 Nfissel, D.R., Serotonin and serotonin immunoreactive neurons in the nervous system of insects, Prog. Neurobiol., 30 (1988) 1-85. 21 Orchard, I., Martin, R.J., Sloley, B.D. and Downer, R.G.H., The association of 5-hydroxytryptamine, octopamine, and dopamine with the intrinsic (glandular) lobe of the corpus cardiacum of Locusta migratoria, Can. J. Zool.. 64 (19861 271-274. 22 Parent, A., The anatomy of serotonin-containing neurons across phylogeny. In B.L. Jacobs and A. Gelperin (Eds.), Serotonin Neurotransmission and Behavior, MIT Press, Cambridge, 1981, pp. 3-34. 23 Pentreath, V.W., Berry, M.S. and Osborne, N.N., The serotonergic cerebral cells in gastropods. In N.N. Osborne (Ed.), The Biology of Serotonergic Transmission, Wiley, Chichester, 1982, pp. 457-514. 24 Rude, S., Monoamine-containing neurons in the central nervous system and peripheral nerves of the leech, Hirudo medicinalis, J. Comp. Neurol. , 16 (1969) 349-372. 25 Shipper, J. and Tilders, F.J.H., A new technique for studying specificity of immunocytochemical procedures: specificity of serotonin immunostaining, J. Histochem. Cytochem., 31 (1983) 12-18. 26 Sukhdeo, M.V.K., Hsu, C.S., Thompson, C.S. and Mettrick, D.F., Hymenolepis diminuta: behavioural effects of 5-hydroxytryptamine, acetylcholine, histamine and somatostatin, J. Parasitol., 70 (1984) 682-688. 27 Tandler, C.J., Bursco, A., Peressini, S. and Pecci Saaredra, J., Further evidence for the specificity of anti-5HT (serotonin)-like antisera in immunocytochemistry, Histochem., 85 (1986) 67-72. 28 Webb, R.A. and Mizukawa, K., Serotonin-like immunoreactivity in the cestode Hymenolepis diminuta, J. Comp. Neurol.. 234 (1985) 431-440. 29 Welsh, J.H., Pylogenetic aspects of the distribution of biogenie amines. In J.J. Blum (Ed.), Biogenic Amines as Physiological Regulators, Prentice-Hall Inc., Englewood Cliffs, New Jersey, 1970, pp. 75 94. 30 Welsh, J.H. and Williams, L.D., Monoamine-containing neurons in planaria, J. Comp. Neurol.. 138 (1970) 103-116. 31 Willcockson, W.S. and Hillman, G.R., Drug effects of the 5-HT response of Schistosoma mansoni, Comp. Biochem. Physiol., 77C (1984) 199-203.
57
Elsevier BRE 13997
Immunohistochemical and electrochemical detection of serotonin in the nervous system of Fasciola hepatica, a parasitic flatworm Suzanne C. Sukhdeo and Michael V.K. Sukhdeo Department of Zoology, University of Toronto, Toronto, Ont. (Canada) (Accepted 11 May 1988)
Key words: Serotonin; Immunohistochemistry; Cerebral ganglion; Bilateral symmetry
The head region of the trematode parasite Fasciola hepatica contains 3.47 _+0.42 pmol/mg wet wt. of serotonin as measured by highperformance liquid chromatography coupled to electrochemical detection. The head region includes the cerebral ganglia, the transverse commissure and associated nervous tissue that innervates the musculature of the oral sucker, pharynx and body wall. Tissue from the tail, which contains little nervous innervation, has approximately 20 times less serotonin (0.18 + 0.01 pmol/mg wet wt.). Immunohistochemistry was used to identify serotonin-like immunoreactive cells. Bipolar and multipolar cell bodies in the cerebral ganglia show serotonin-like immunoreactivity. Also evident are serotonin-like immunoreactive processes in the neuropile and in the transverse commissure that connects the ganglia, and immunoreactive peripheral bipolar cell bodies innervating the musculature of the pharynx and body wall. The cell bodies containing serotonin are organized in bilateral symmetry with homologous cell bodies and processes represented in each ganglion and on both sides of the pharynx.
INTRODUCTION The Phylum Platyhelminthes (flatworms) contains first seen in the Phylum Platyhelminthes22'29 and the ly symmetrical metazoans and the first animals to possess 'true' brains 5. Brains and bilateral symmetry are features that are retained in subsequent evolutionary development and these organisms occupy a crucial position at the junction of vertebrate and invertebrate phylogenetic radiation I. Serotonin has been recovered from the tissues of several flatworms 3"7"19"22"29 where it is believed to exert biochemical and hormonal effects on carbohydrate metabolism 16'18. A n u m b e r of studies using Falck-Hillarp, radioautographic and immunochemical techniqueg have also localized serotonin in cells of the central and peripheral nervous systems of flatworms 2'8"12"28'3°. There is some evidence that serotonin may function as an excitatory neurotransmitter and although the effects are somewhat inconsistent26, serotonin does increase the motility of several flat-
worm species 15"tT'26`3j. The excitatory effect is believed to be mediated by the peripheral nerves because it is not affected by decerebration 17'26. The functions of the n e u r o n s with serotonin-like i m m u n o reactivity that are found in the cerebral ganglia of flatworms 1°12"13are unclear.
Fasciola hepatica (Trematoda) is a large parasitic flatworm that is a feasible candidate for electrophysiological studies because of its size and the ease of m a i n t e n a n c e in the laboratory. The purpose of this study was: (1) to determine if endogenous serotonin is present in the tissues of F. hepatica using a sensitive and specific assay based on high-performance liquid chromatography; and (2) to map the distribution of serotonin-like neurons in the head region of this organism using an immunohistochemical technique. MATERIALS AND METHODS The infective metacercarial stages of F. hepatica were purchased from a commercial supplier (Bald-
Correspondence: S. Sukhdeo, Department of Zoology, University of Toronto, Toronto, Ont. M5S 1A1, Canada. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
58 win Enterprises, Oregon) and 15-20 metacercariae were given to Wistar rats (Woodlyn Labs., Guelph, Ont.) by intragastric inoculation. Adult worms were collected from the bile ducts of rats infected 4 - 6 months previously and placed in a buffered ringer solution for dissection. The head region, approximately 2 mm in length (containing the oral sucker, pharynx, paired cerebral ganglia, transverse commissure and mesenchyme), was separated from the body and used for immunohistochemistry and HPLC determinations. F, hepatica is an acoelomate and all the organs are embedded in mesenchyme. It is impossible to distinguish between different tissues in vivo and the selective dissection and removal of the cerebral ganglia is technically unfeasible. Two preparations of the head region were made for immunofluorescence experiments, a dorsal and a ventral preparation. In the dorsal preparation, the dorsal tegument was carefully dissected away from the head to expose the underlying tissues to the immunochemical solutions. In the ventral preparation, the ventral tegument was removed.
Electrochemical detection of serotonin Serotonin was assayed by high-performance liquid chromatography (HPLC) using the electrochemical detection procedure of Orchard et al. el. The head regions of the organisms were used for the HPLC assay but the dorsal and ventral tegumental coverings were left intact. Tissue from the tail region, of similar size and weight to the head region, were also examined. The tail region contains little nervous innervation. The protocol for the preparation of the tissue for HPLC was modified from the procedure described by Orchard et al. 21. Briefly, 3 heads or 3 tails were pooled, weighed (approximate weight 8 mg) and placed in 30/A of ice cold 0.2 N perchloric acid and frozen a t - 2 0 °C. The tissue was thawed and 175/~1 of HPLC buffer was added. The tissue was sonicated in the mixture for 30 s, centrifuged at 8800 g for 5 - 7 minutes and then filtered through a 0.22 ktm nylon filter. A 100/~1 aliquot of the filtered supernatant was injected onto a Brownlee RP-18 Spheri-5 HPLC column (4.6 mm x 22 cm). The detection of the eluted compounds was done electrochemically using an ESA model 5100 A detection system coupled to a model 5010 dual coulometric detector. Serotonin was
detected on the second detector set at 0.35 V with the first detector set at 0.1 V acting as a screen. Serotonin was quantified using the external standard method incorporated into an SP4700 integrator. The tissues for each replicate (3 heads or 3 tails per replicate) were spiked with serotonin to confirm the identity of the oxidizable substance and to check for losses.
Serotonin immunohistochemistry The head preparations were fixed for 40 min at room temperature in 2% paraformaldehyde in Millonigs buffer (pH 7.4). After fixation, the preparations were washed for 3 - 4 h in phosphate-buffered saline (PBS) (pH 7.2) which contained 0.9% NaCI. The preparations were then incubated for 1 h in PBS containing 4% Triton X-100, 2% bovine serum albumin and 2% normal goat serum. The anti-serotonin antiserum (lmmuno Nuclear Corp., Stillwater, MN) was diluted 1:1000 in PBS containing 2% bovine serum albumin, 0.4% Triton X-100 and 2% normal goat serum and incubated at 4 °C for 18 h. The preparations were then incubated in the diluted antiserum for 48 h at 4 °C with constant agitation. After antibody incubation, the preparations were washed for 3-4 h with PBS and incubated overnight at 4 °C with fluorescent isothiocyanate-labelled sheep anti-rabbit IgG (Daymar Labs., Toronto, Ont.) diluted 1:200 in 10% normal goat serum in PBS. The preparations were then washed for 2-3 h in PBS and mounted in 5% n-propylgallate made up in glycerol (pH 7.3) in well-slides. The preparations were examined for serotoninlike immunoreactivity with an epi-illumination fluorescent microscope. The data on the distribution of serotonin-like immunoreactivity are based on 6 dorsal and 6 ventral replicates. In addition, 7 control replicates were done to test for the antiserum specificity. The control preparations were incubated with serotonin antiserum that had been preabsorbed with 1 mg/ml serotonin or 100 #g/ml serotonin conjugated to bovine serum albumin (lmmuno Nuclear Corp., Stillwater, MN) for 18 h at 4 °C. RESULTS
Electrochemical detection of serotonin The head region of F. hepatica, which includes the cerebral ganglia and associated nervous tissue, the
59 oral s u c k e r
and
pharynx,
contains
3.47
+
0.42
Cerebral ganglia
p m o l / m g wet wt. of s e r o t o n i n (n = 4). T h e tail re-
N e u r o n s and their processes that w e r e f o u n d to be
gion, which d o e s not c o n t a i n significant n e r v o u s in-
consistently i m m u n o r e a c t i v e are s u m m a r i z e d in c o m -
n e r v a t i o n , has a p p r o x i m a t e l y 20 times less s e r o t o n i n
posite drawings of the dorsal and v e n t r a l p r e p a r a -
(0.18 + 0.01 p m o l / m g wet wt; n = 4).
tions (Fig. 1). All staining was absent in c o n t r o l p r e p -
. .... :::::::::::::::::::::::::: I
•
t
I
%
\;
O.S.~ is
~"
',,
f
o.s.
r/ / !
Nf
I ~l
,%t Ph
!
//
GOb:
~
/
go
Z
eo
/ r
st
.. .:
-,.44
P.!.
I
't!
I I
# ! t
#
t !
-(
A.
B.
/
'
°
Fig. 1. Serotonin-like immunoreactive cells and processes in the head region of Fasciola hepatica. These diagrams represent composites of camera lucida drawings of cells that reacted consistently with the serotonin antiserum. Five cerebral ganglion cell bodies (GCb) were observed from the dorsal (A) view and 3 could be seen in the ventral (B) view. The ganglion cell bodies extend processes into their respective neuropile area (Nf), anterior to innervate the oral sucker (O.S.), posterior to innervate the pharynx (Ph) and contralateral processes through the dorsal transverse commissure (COM, A). Peripheral cell bodies (Cb) innervating the pharynx (Ph) also send contralateral nerve processes over the dorsal (A) and ventral (B) surfaces of the pharynx.
60
Fig. 2. Whole mount preparations showing serotonin-like immunoreactive cells and nerve processes in the head region of F. hepatica. A: dorsal view of the head showing two immunofluorescent areas which represent the paired cerebral ganglia (Curved arrows) on either side of the junction between the oral sucker (OS) and the pharynx (P), (E = egg), scale bar = 20/~m; B: higher magnification of one cerebral ganglion showing two immunoreactive ganglion cell bodies in focus (Cb) and a meshwork of immunoreactive nerve fibers of the neuropile (curved arrows). One process (arrowhead) extends anterior to innervate the oral sucker, scale bar = 10 urn; C: a bipolar ganglion cell with stained nucleus (N) and process (arrowhead) extending towards the oral sucker, scale bar = 10gtm; D: two cell bodies (arrows) situated on either side of the pharynx sending contralateral processes (arrowhead) over the pharynx midline (PM, large arrow), scale bar = 2t)/~m.
61 arations where the serotonin antiserum was pre-absorbed with serotonin-conjugated to BSA. Homologous bilaterally symmetrical neurons showing serotonin-like immunoreactivity occur in both the dorsal and ventral aspects of the cerebral ganglia (Figs. 1 and 2A). Five serotonin neurons occur in each ganglion and 3 of these can be seen from the ventral aspect (Figs. 1 and 2B). The majority of these neurons appear spindle-shaped with longitudinal axes of 25-30 ~m (Figs. 1 and 2B). The neurons are a mixture of bipolar, multipolar and monopolar types with nerve processes that extend out of the ganglia to innervate the musculature of the oral sucker and pharynx (Figs. 1 and 2D). Neurites also extend into the neuropile of the ganglia and the immunoreactive neurons of the ganglia surround a core of finely stained processes in the neuropile (Figs. 1 and 2B). Six to 10 serotonin immunoreactive processes connect the ganglia via the transverse commissure (Fig. 1A).
fore, one may be reasonably confident that the cell bodies and processes described in this study do contain serotonin. The serotonin-like immunoreactive neurons in the cerebral ganglia of F. hepatica are bilaterally symmetrical with 5 neurons occupying similar locations in each ganglion. Some of the nerve processes from these neurons clearly extend to the contralateral ganglion via the transverse commissure. The bilateral symmetry of homologous serotonin immunoreactive neurons that extend contralateral processes is also seen in the peripheral neurons innervating the pharynx. In this study of F. hepatica and in other studies of flatworms, many of the serotonin-containing neurons appear to be motor neurons that are responsible for innervation of the musculature of the digestive tract and body wall 3°. Although not fully substantiated by electrophysiological data, this preferential localization of serotonin in the motor neurons of invertebrates has been reported by several investigatorsll'22
Peripheral immunoreactive cells
We believe that the bilaterally symmetrical distribution of serotonergic neurons may have significant functional implications in the evolution of the serotonin system. Serotonin has been recovered from the tissues of representatives of most animal phyla 6A4, but the association of serotonin with nervous tissue is first seen in the Phylum Platyhelminthes 22'29 and the occurrence of serotonin neurons coincides with the development of bilateral symmetry. The bilaterally symmetrical distribution of ganglionic serotonergic neurons described in this study of F. hepatica is not restricted to this flatworm species. The Falck-Hillarp technique shows bilaterally symmetrical serotonin cells with contralateral processes in the cerebral ganglia of several free-living flatworms (Turbellaria) 3° and a bilateral symmetry of serotonergic structures in the ganglia of parasitic flatworms (Trematoda and Cestoda) 2'15. This pattern of bilateral symmetry and contralateral processes of serotonergic neurons is conserved in the higher invertebrate phyla and is seen, for example, in the giant serotonin-containing cells in ganglia of molluscs 23 and in the serotonergic neurons in the brains and ganglia of many annelid 23"24 and insect species 4,z°. We suggest that the pattern of homologous bilaterally symmetrical ganglionic neurons, that extend contralateral processes, reflects a primitive function of coordination of muscular activi-
There are numerous small bipolar neurons (10-15 ~tm longitudinal axis) (Fig. 2C) showing serotoninlike immunoreactivity that are not associated with the cerebral ganglia. Many of these neurons innervate the pharynx where they are organized in bilateral symmetry and typically send contralateral processes around the pharynx (Figs. 1 and 2D). Similar bilaterally symmetrical, bipolar neurons with their neurites oriented to the longitudinal axis of the worm innervate the musculature of the body wall of the animal. DISCUSSION The serotonin antiserum used in the present study has been used in many immunochemical studies and is reported to have high specificity with little crossreactivitf 5,27. This immunohistochemical staining in itself does not constitute chemical identification of serotonin; however, pre-absorption of the antiserum with serotonin conjugated to bovine serum albumin abolishes all staining. In addition, HPLC coupled to electrochemical detection (an accurate and sensitive assay 9) identifies the presence of serotonin in the tisrues of F. hepatica and suggests that this amine is con:entrated in the head region of this organism. There-
62 ty t h a t e v o l v e d in t h e first b i l a t e r a l l y s y m m e t r i c a l organisms.
a n d A n g e l a L a n g e for p e r f o r m i n g t h e H P L C a n a l y s i s and assistance with the immunohistochemistry. This s t u d y was s u p p o r t e d by a n O n t a r i o G r a d u a t e S c h o l -
ACKNOWLEDGEMENTS
a r s h i p to S.C.S. a n d N S E R C
G r a n t A 4 6 6 7 to D . F .
Mettrick. T h e a u t h o r s w o u l d like to t h a n k D r s . Ian O r c h a r d
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