Cell Tissue Res. 198, 95-104 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
Autoradiographic Detection of Indolamine and Catecholamine Neurons in the Nervous System of Owenia fusiformis (Polychaeta, Annelida) J. Coulon and R. Bessone* Laboratoire de Morphogrn+seAnimale, Drpartement de Biologic Cellulaire et Molrculaire, MarseilleLuminy; Institut de Neurophysiologieet Psychophysiologie,Drpartement de Neurobiologie Cellulaire, Marseille, France
Summary. Light- and electron-microscopic autoradiographic studies were carried out on Owenia to detect selectively catecholaminergic and indolaminergic neurons at two appropriate body levels according to the regenerative properties of this annelid, i.e. in the 1st and the 4th abdominal segments. Autoradiographically, intense aH-5-hydroxytryptamine accumulation is seen in the 1st abdominal segment, and a less intense autoradiographic reaction in the 4th segment. Roughly, a similar difference in the distribution of the label is seen at both levels following 3H-noradrenaline administration. These observations, confirmed by a quantitative uptake analysis, suggest the presence of catecholaminergic and indolaminergic neurons in Owenia. Key words: Monoaminergic neurons - Autoradiography - Annelid - Owenia f u s i f ormis (Polychaeta).
The presence of endogenous biogenic amines in invertebrates is well documented (Gerschenfeld, 1973). In Nephtydidae, Nereidae and Glyceridae (Polychaeta), serotonin and noradrenaline have been demonstrated by histofluorescence (Clarck, 1966; Dhainaut-Courtois, 1970) and biochemical assays (Manaranche and l'Hermite, 1973). Since monoaminergic neurons are capable of taking up and retaining selectively their neurotransmitters, they can be detected by autoradiography following administration of the respective labeled transmitter (Aghajanian, 1966; Iversen, 1974). This approach has been used in invertebrates to detect selectively catecholaminergic and indolaminergic neurons at light microscopic (Coggeshall, 1974; G o h and Davey, 1976) and ultrastructural levels (Dhainaut-Courtois and Dhainaut, 1976). Send offprint requests to: Dr. J. Coulon, Institut de Biologic Cellulaire, LA n~
Centre Universitaire de Marseille-Luminy,Route L. Lachamp, 13288 Marseille Cedex 2, France * The authors thank Dr. A. Calas for useful discussions and critical reading of the manuscript, and G. Monty for his technical assistance. The investigationscited in the present paper were supported by a research grant n~ from the INSERM
0302-766X/79/0198/0095/$02.00
96
J. Coulon and R. Bessone
In Owenia, a species t h a t has been t h o r o u g h l y studied for its regenerative p r o p e r t i e s ( T h o u v e n y , 1967), no d a t a are available c o n c e r n i n g m o n o a m i n e r g i c i n n e r v a t i o n . H o w e v e r , the m o r p h o l o g i c a l a p p e a r a n c e o f dense core vesicles present in t e r m i n a l s a n d n e u r o n s o f the ventral c o r d m i g h t indicate t h a t they are related to biogenic a m i n e s ( D a h l s t r 6 m , 1972; Pellegrino de I r a l d i et al., 1971). F u r t h e r m o r e , in this species, d a i l y v a r i a t i o n s in n u m b e r o f e l e m e n t a r y vesicles in a x o n s o f the nerve c o r d have been f o u n d d u r i n g r e g e n e r a t i o n ( C o u l o n et al., 1976). A f t e r cephalic a m p u t a t i o n , the regenerative p r o p e r t i e s o f Owenia decrease following an a n t e r o - p o s t e r i o r g r a d i e n t (Thouveny, 1967). A t the level o f the 1st a b d o m i n a l segment r e g e n e r a t i o n is always complete, while at the level o f the 4th a b d o m i n a l segment it has never been observed, a l t h o u g h a s t i m u l a t i o n o f resting cells takes place after t r a u m a ( C o u l o n a n d Marilley, u n p u b l i s h e d results). It has been suggested t h a t m o n o a m i n e n e u r o t r a n s m i t t e r s m i g h t p l a y an i m p o r t a n t role in regenerative processes in invertebrates ( C h a p r o n a n d C h a p r o n , 1972; F r a n q u i n e t et al., 1976; C o u l o n a n d Marilley, 1978). Thus, it was the aim o f the p r e s e n t s t u d y to detect the presence o f i n d o l a m i n e a n d c a t e c h o l a m i n e n e u r o n s at light a n d electron m i c r o s c o p i c levels, t a k i n g a d v a n t a g e o f the r e u p t a k e c a p a c i t y o f these neurons.
Materials and Methods
Autoradiography The 1st and 4th abdominal segments from specimens of Oweniafusiformis were incubated in sea water for 20 to 60min with: a) 3H-5-hydroxytryptamine (5-HT) (Amersham/Searle, specific activity 21 Ci/mM, concentration 7.10- SM); b) 3H-noradrenaline (NA) (CEA/Saclay, specific activity 20 Ci/mM, concentration 10-7M). In some cases the labeled transmitters were supplemented with unlabeled ones or with a drug: a) 3H-5-HT (10-TM) and cold NA (10-6M). b) 3H-5-HT (10-TM) and fluoxetin (2.10-6M). c) 3H-5-NA (10-7M) and cold 5-HT (10-6M). The radioactive tracers were preserved from chemical oxidation by addition of 0.002 700ascorbic acid. After incubation, all samples were washed for 15 min in sea water with the corresponding nonradioactive transmitter (10-4 M), then fixed in 2 ~ glutaraldehyde in cacodylate buffer and sea water. For light microscopic autoradiography, the pieces were embedded in paraffin. The sections (7 pin thick) were coated with Ilford K5 emulsion diluted 1 : 1 with distilled water, then exposed for 10 days. For electron microscopic autoradiography, the samples were postfixed in osmium and embedded in Araldite. The ultrathin sections were coated with Ilford L4 emulsion diluted 1 : 4 (Larra and Droz, 1970), exposed for 21 days, then developed in Kodak D 19b. The grids were observed with a HitachiHU 12 electron microscope.
Quantitative Uptake A study of quantitative uptake was performed according to Ternaux et al. (I 977). Portions of the nerve cord at different body levels of seven pooled animals were homogenized in buffered artificial sea water (in mM : Tris-Hcl pH 7.5,10; NaC1, 494; CaC12,10; K C1,10; MgSO4, 30; Mg C12,20) with ascorbic acid and sucrose, and transferred to Eppendorf microtubes containing 400 ~tlof the same medium. 3H-5-HT (7.10-SM) or aH-NA (7.10-aM) was added and incubation was performed for 8min at 25~ The reaction was stopped by adding 400~tl of non-radioactive sea water and incubating at 0~ After
Indolamine and Catecholamine Neurons of Oweniafusiformis
97
centrifugation for 4min at 9,800 x g, the pellet was washed with 400~tl of artificial sea water, and suspended in 200 ~tlof water containing Triton X-100 (0.5 %) for scintillation counting. An aliquot of the homogenate was sampled for protein determination. Controls were obtained by incubating samples at 0~ Results In Owenia, the medio-ventral nerve cord is in an intra-epithelial position. The nerve fibers are located on the external side of the basal lamina, and the perikarya are scattered a m o n g epidermal cells. Axonal processes extending to the dorsal side are found laterally between the basal lamina and the epidermis (Fig. 1) (Coulon and Thouveny, 1974).
In vitro Incubation in 3H-5-HT The time course of labeling studied for periods varying from 20 to 60 min gives identical autoradiographic reactions. Following 3H-5-HT administration, in cross sections at the level of the 1st abdominal segment a diffuse autoradiographic reaction is observed on all structures; it is more pronounced on the epidermal cells. In contrast, dense and selective silver grain accumulations are located in the central part of the nerve cord and in its lateral (Figs. 1,2) and dorsal processes. In this respect the serial sections show a uniform pattern of the autoradiographic reaction. At the level of the 4th abdominal segment, there is a decrease in the selective reaction in the nerve cord and the axonal processes (Fig. 3). Addition of cold NA at a tenfold concentration produces the same autoradiographic pattern at both levels, i.e. the 1st (Fig. 4) and the 4th abdominal segments. On the other hand, after incubation in aH-5-HT and fluoxetin, a selective autoradiographic reaction on the nerve cord and lateral fibers is no longer found, although the diffuse reaction and a more pronounced one on the adjacent epidermal cells persist (Fig. 5). The labeled structures can be identified by means of electron microscopic autoradiography. Silver grain accumulations located in juxtaposition to the basal lamina at the level of the ventral nerve cord or at the level of the lateral and dorsal axonal processes correspond to axonal varicosities containing dense core vesicles (60-90 nm in diameter) (Fig. 9). In the central part o f the nerve cord, silver grains are seen mainly on the axonal fibers. At the periphery of the nerve cord, some perikarya show an autoradiographic reaction over the nucleus and the cytoplasm (Fig. 10). Study of the quantitative uptake by scintillation counting shows a decrease of the radioactivity in the nerve cord following an antero-posterior gradient. The maximal value is obtained at the level of the 1st segment, while minimal values are seen at the level of both the 3rd and 4th segments (Table 1).
In vitro Incubation in 3H-NA The autoradiographic reactions are similar after an incubation time varying from 20 to 60 min.
Figs. 1-3. Autoradiographs of cross sections of Owenia following incubation with aH-5-HT for 30 min (dark field illumination, Figs. 2 and 3). Fig. 1. Medio-ventral position of the nerve cord (nc) in epidermis (e) and the lateral axonal processes (ap). Note the intense labeling in the central part of the nerve cord; brn basal lamina (membrane); m muscle. • 300. Fig. 2. First abdominal segment. Three types of A R G reactions are observed (see text). Note the selective labeling in the central part of the nerve cord (nc) and along the lateral axonal processes (arrows). x 300. Fig. 3. Fourth abdominal segment. The intensity of the labeling decreases in the ventral nerve cord (nc). x 300
Indolamine and Catecholamine Neurons of Oweniafusiformis
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Figs. 4 and 5. Dark field autoradiographs o f cross sections o f Owenia at the level o f the I st abdominal segment following 3H-5-HT incubation with cold NA (Fig.4) or fluoxetin (Fig.5). Fig.4. The distribution o f the labeling resembles that observed in Fig. 2. x 300. Fig. 5. Absence of silver grain accumulation over the nervous structures, the persistence o f a diffuse reaction and a more pronounced accumulation in the epidermal cells. • 300
Table 1. Quantitative fluctuations o f 3H-5-HT uptake and 3H-NA uptake in various areas of the nerve cord. Controls were obtained by incubating samples at 0~ (Mean values + standard error) Topography
Uptake of 3H-5-HT ( x 10-4 ~tCi/mg Prot.)
Uptake o f 3H-NA ( x 10-4 ~tCi/mg Prot.)
First abdominal segment Second abdominal segment Third abdominal segment Fourth abdominal segment Controls
34.17 + 26.3 + 17.5 + 17.0 + 6.9 +
6.79 ___0.48 5.80+ 0.61 4.69 ___0.22 5.23 + 0.73 1.4 + 0.09
3.82 5.86 0.71 1.0 0.21
Figs. 6-8. Dark field autoradiographs of cross sections of Owenia following 3H-NA administration alone (Figs. 6, 7) or with cold 5-HT (Fig. 8). Fig. 6. First abdominal segment. Silver grains are mainly located close to the basal lamina of the nerve cord. The axonal processes are poorly labeled, x 300. Fig. 7. Fourth abdominal segment. The distribution of the labeling resembles that observed in the 1st abdominal segment, x 300. Fig. 8. Fourth abdominal segment. Addition of cold 5-HT to the incubation medium does not alter the distribution of the label
Figs. 9and 10. Electron microscopic autoradiographs after incubation in 3H-5-HT for 30min. Fig. 9. Labeled varicosity containing numerous dense core vesicles (arrows) near an uniabeled one (*). Varicosities are surrounded by profiles of glial cells (gc). x 11,300. Fig. 10. Cell body showing positive reaction on nucleus and cytoplasm. • 20,000 Figs. 11 and 12. Electron microscopic autoradiographs after incubation in 3H-NA for 30min. Fig. 11. Uptake of aH-NA by axonal varicosities containing clear and dense core vesicles (arrows). • 16,500. Fig. 12. Labeled cell body. Note the presence of dense core vesicles, x 16,500
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At the level of the 1st abdominal segment, the 3H-NA reaction is less intense than the 3H-5-HT reaction. However, a selective labeling is seen in the central part of the nerve cord and near the basal lamina (Fig. 6). A reduced number of grains is usually observed in the lateral and dorsal fibers. The 4th abdominal segment exhibits principally the same distribution of the label (Fig. 7). In worms incubated with 3H-NA and tenfold concentrated cold 5-HT, a similar distribution of label is found at the level of both the 1st and 4th abdominal segments (Fig. 8). Ultrastructurally, labeled axonal varicosities contain vesicles with varying-sized dense cores (50-70 nm in diameter) (Fig. 11). Some cell bodies scattered among epidermal cells are also labeled (Fig. 12). Quantitative distribution of the radioactive material following 3H-NA incubation is comparable at the first two segments. Moreover, a slight decrease is found at the level of the 3rd and 4th segments (Table 1).
Discussion
Radioactivity observed in amine-containing tissues by autoradiography and by scintillation counting may be due to the radioactive amines and/or their metabolites. Monoamine oxidase is relatively inactive in invertebrates (Hiripi and Salanki, 1971), but a recent biochemical study has shown the formation of sugar conjugates after 1 to 15 h following in vitro administration of labeled serotonin in Aplysia (Goldman and Schwartz, 1977). Thus it is probable that after a shorter incubation time, the major part of radioactivity arises from the amine itself, which was protected from chemical oxidation by an antioxidant. Concerning autoradiographic (ARG) studies, three types of reactions should be regarded separately: 1) The diffuse, non-selective ARG reaction is usually observed after incubation with each amine. This type corresponds to an artefactual retention by glutaraldehyde of the free or weakly bound monoamines and their metabolites and indicates tracer penetration into the tissue (Descarries and Droz, 1970). 2) The more pronounced reaction, which can be seen on some structures of the epidermis mainly in the sagittal part of the first abdominal segment, might be attributed to a retention capacity of the epidermis, which varies following an antero-posterior gradient (Thouveny, unpublished results). 3) The intense and selective silver grain accumulation indicates the sites where the tracer has been taken up and retained. Published data obtained in vertebrates as well as in invertebrates indicate that the intensely labeled structures following 3H-5-HT and 3H-NA administration are monoaminergic neurons (Descarries and Dupin, 1974; Goh and Davey, 1976, Bessone et al., 1978). Furthermore, following in vitro incubation with the labeled transmitters at low concentrations (10-7M and below), autoradiography and analysis of quantitative uptake allow a specific detection of serotoninergic or catecholaminergic neurons in vertebrates (Shaskan and Snyder, 1970) as well as in invertebrates (Bessone et al., 1978). Based on this general knowledge, our results in Owenia suggest the existence of different kinds of monoaminergic neurons:
Indolamine and Catecholamine Neurons of Oweniafusiformis
103
1) Different autoradiographic patterns are observed following 3H-5-HT and 3H-NA incubation, respectively. 2) Addition of unlabeled NA or 5-HT to the incubation medium does not change the distribution of silver grains in tissues incubated previously with 3H-5-HT or 3H-NA. 3) The absence of an intense A R G reaction following addition of fluoxetin (a selective inhibitor of 5-HT uptake by serotoninergic neurons; H a m o n , 1975) appears to prove the existence of serotoninergic neurons. These results are confirmed by a study of the quantitative uptake and are in agreement with biochemical assays of 5-HT performed in another polychaete, Glycera (Manaranche, 1975). On the other hand, neurons labeled following 3H-NA administration may be defined as catecholaminergic (CA) since CA neurons have the ability to take up and retain nonspecifically noradrenaline or dopamine tracers (Descarries and Droz, 1970). Morever, they are probably noradrenergic, since, in comparison to noradrenaline, a very weak concentration of dopamine has been found by biochemical assays in another polychaete annelid (Manaranche, 1975). At the ultrastructural level, the structures intensely labeled following 3H-5-HT or 3H-NA administration correspond to those studied light microscopically, and therefore can be considered as serotoninergic or noradrenergic varicosities. The aspect of their granular vesicles appears to differ ultrastructurally; however, in other species, a uniform type of vesicle has been found in tissues storing different amines (Pellegrino de Iraldi et al., 1971). The analysis of the quantitative uptake indicates the predominance of 5-HT innervation in the ventral nerve cord of Owenia; this is in agreement with biochemical determinations of 5-HT in other invertebrates (Gerschenfeld, 1973; Manaranche, 1975). Moreover, the decrease in neurotransmitter uptake in the 4th abdominal segment might be related to the decrease in the density of the monoaminergic innervation and/or to a modified activity of these fibers. It has to be proven whether this peculiarity m a y be related to the loss of the regenerative capacity of the 4th abdominal segment.
References Aghajanian, G.K., Bloom, F.E., Lowell, R.A., Sheard, M., Freedman, D.X.: The uptake of 5hydroxytryptamine-ZH from the cerebral ventricles: autoradiographic localization. Biochem. Pharmacol. 15, 1401-1403 (1966) Bessone, R., Segu, L., Calas, A.: Radioautographic identification of serotoninergic neurons in Aplysia. Experentia 34, 1038-1039 (1978) Chapron, C., Chapron, J.: Influence des amines biogrnes et de leurs inhibiteurs sur la rrgrn~ration. Etude chez l'Ann~lide Eiseniafoetida. C.R. Acad. Sci., Paris 274, D, 412-414 (1972) Clarck, M.E.: Histochemical localization of monoamines in the nervous system of the polychaete Neptys. Proc. Roy. Soc. B. 165, 308-325 (1966) Coggeshall, R.E.: Autoradiographic and chemical localization of 5-hydroxytryptamine in identified neurons in the leech. Anat. Rec. 172, 489-498 (1972) Coulon, J., Marilley, M.: Fluctuations of adenylate cyclase activity during anterior regeneration in Oweniafusiformis (PolychaeteAnnelid). J. Embryol. Exp. Morph., 48, 73-78 (1978) Coulon, J., Thouveny, Y.: Etude ultrastructurale de la d~diffrrenciation cellulaire dans la rbg~n~ration ant~rieure de l'Ann61ide: Oweniafusiformis. Ann. Embryol. et Morph. 7, 61-79 (1974)
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Coulon, J., M arilley, M., Thouveny, Y.: Variations j ournali6res du taux des s6cr6tions nerveuses chez Oweniafusiformis. Persistance d'un rythme journalier dans les tissus en culture. C.R. Acad. Sci., Paris 283, D, 1535-1538 (1976) Dahlstr6m, A.: Aminergic transmission. Introduction and short review. Brain Res. 62, 441-460 (1972) Dainhaut-Courtois, N.: Etude en microscopie 61ectronique et en fluorescence des m6diateurs chimiques du syst6me nerveux des Nereidae (Ann6lides Polych6tes). Z. Zellforsch. 126, 90-103 (1970) Dainhaut-Courtois, N, Dhainaut, A.: Gamma-aminobutyric acid and 5-hydroxytryptamine in the nervous system of an annelid worm (Nereis diversicolor). J. Microsc. Biol. Cell 27, 261-266 (1976) Descarries, L., Droz, B.: Intraneural distribution of exogenous norepinephrine in the central nervous system of the rat. J. Cell Biol. 44, 385-399 (1970) Descarries, L., Dupin, J.C.: Retention of noradrenaline-3H in brain and preferential extraction of labeled metabolites by glutaraldehyde fixation. Experientia 30, 1164-1165 (1974) Franquinet, R., Stengel, D., Hanoune, J.: The adenylate cyclase system in a fresh-water planarian (Polycelis tenius IIJIMA). Comp. Biochem. Physiol. 53B, 329-333 (1976) Gerschenfeld, H.M.: Chemical transmission in invertebrate central nervous system and neuromuscular junctions. Physiol. Rev. 53, 1-119 (1973) Goh, S.L., Davey, K.G.: Selective uptake of noradrenaline, DOPA, and 5-hydroxytryptamine by the nervous system of Phocanema decipiens (Nematoda): a light autoradiographic and ultrastructural study. Tissue and Cell 8, 421-435 (1976) Goldman, J.E., Schwartz, J.H.: Metabolism of aH-serotonin in the marine mollusc, Aplysia californica. Brain Res., 136, 77-88 (1977) Hamon, M.: Biosynth6se de la s+rotonine dans le syst+me nerveux central. Quelques aspects de sa r6gulation. Th6se Dt es-sci. Nat. Paris VII (1975) Hiripi, L., Salanki, J.: The role of monoamine-oxydase in the inactivation of serotonin in the nervous system and other tissues of Anodonta cygnea. Ann. Biol. tihany. 38, 31-38 (1971) Iversen, L.L.: Uptake mechanisms for neurotransmitter amines. Biochem. Pharmacol. 7, 1927-1935 (1974) Larra, F., Droz, B.: Techniques radioautographiques et leur application ~ l'+tude du renouvellement des constituants cellulaires. J. Microscopic 9, 845-880 (1970) Manaranche, R.: Contribution fi la connaissance du syst6me nerveux de Glycera convoluta (K.) Ann61ide Polych~te. Th~se Dt es-sci. Nat. Paris (1975) Manaranche, R., L'Hermite, P.: Etude des amines biog6nes de Glycera convolute K. (Ann61ide Polych6te). Z. Zellforsch. 137, 21-36 (1973) Pellegrino de Iraldi, A., Gueudet, R., Suburo, A.M.: Differentiation between 5-hydroxytryptamine and cateeholamine in synaptic vesicles. Progr. Brain Res. 34, 161 170 (1971) Shaskan, E.G., Snyder, S.H.: Kinetics of serotonin accumulation into slices from rat brain: relationship to catecholamine uptake. J. Pharmacol. Exp. Therap. 175, 404-418 (1970) Sotelo, C.: The fine structural localization of norepinephrine-aH in the substantia nigra and area postrema of the rat. An autoradiographic study. J. Ultrastruct. Res. 36, 824-841 (1971) Ternaux, J.P., Hery, F., Bourgoin, S., Adrien, J., Glowinski, J., Hamon, M.: The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat. Brain Res. 121, 311 326 (1977) Thouveny, Y.: Etude exp6rimentale et biochimique du fonctionnement des syst6mes histog6n6tiques et de la d6diff6renciation cellulaire dans la morphogen6se des Annblides Polych+tes. Th6se Dt es-sci. Nat. Marseille (1967)
Accepted December 10, 1978
Cell and Tissue Research 9 by Springer-Verlag 1979
Autoradiographic Detection of Indolamine and Catecholamine Neurons in the Nervous System of Owenia fusiformis (Polychaeta, Annelida) J. Coulon and R. Bessone* Laboratoire de Morphogrn+seAnimale, Drpartement de Biologic Cellulaire et Molrculaire, MarseilleLuminy; Institut de Neurophysiologieet Psychophysiologie,Drpartement de Neurobiologie Cellulaire, Marseille, France
Summary. Light- and electron-microscopic autoradiographic studies were carried out on Owenia to detect selectively catecholaminergic and indolaminergic neurons at two appropriate body levels according to the regenerative properties of this annelid, i.e. in the 1st and the 4th abdominal segments. Autoradiographically, intense aH-5-hydroxytryptamine accumulation is seen in the 1st abdominal segment, and a less intense autoradiographic reaction in the 4th segment. Roughly, a similar difference in the distribution of the label is seen at both levels following 3H-noradrenaline administration. These observations, confirmed by a quantitative uptake analysis, suggest the presence of catecholaminergic and indolaminergic neurons in Owenia. Key words: Monoaminergic neurons - Autoradiography - Annelid - Owenia f u s i f ormis (Polychaeta).
The presence of endogenous biogenic amines in invertebrates is well documented (Gerschenfeld, 1973). In Nephtydidae, Nereidae and Glyceridae (Polychaeta), serotonin and noradrenaline have been demonstrated by histofluorescence (Clarck, 1966; Dhainaut-Courtois, 1970) and biochemical assays (Manaranche and l'Hermite, 1973). Since monoaminergic neurons are capable of taking up and retaining selectively their neurotransmitters, they can be detected by autoradiography following administration of the respective labeled transmitter (Aghajanian, 1966; Iversen, 1974). This approach has been used in invertebrates to detect selectively catecholaminergic and indolaminergic neurons at light microscopic (Coggeshall, 1974; G o h and Davey, 1976) and ultrastructural levels (Dhainaut-Courtois and Dhainaut, 1976). Send offprint requests to: Dr. J. Coulon, Institut de Biologic Cellulaire, LA n~
Centre Universitaire de Marseille-Luminy,Route L. Lachamp, 13288 Marseille Cedex 2, France * The authors thank Dr. A. Calas for useful discussions and critical reading of the manuscript, and G. Monty for his technical assistance. The investigationscited in the present paper were supported by a research grant n~ from the INSERM
0302-766X/79/0198/0095/$02.00
96
J. Coulon and R. Bessone
In Owenia, a species t h a t has been t h o r o u g h l y studied for its regenerative p r o p e r t i e s ( T h o u v e n y , 1967), no d a t a are available c o n c e r n i n g m o n o a m i n e r g i c i n n e r v a t i o n . H o w e v e r , the m o r p h o l o g i c a l a p p e a r a n c e o f dense core vesicles present in t e r m i n a l s a n d n e u r o n s o f the ventral c o r d m i g h t indicate t h a t they are related to biogenic a m i n e s ( D a h l s t r 6 m , 1972; Pellegrino de I r a l d i et al., 1971). F u r t h e r m o r e , in this species, d a i l y v a r i a t i o n s in n u m b e r o f e l e m e n t a r y vesicles in a x o n s o f the nerve c o r d have been f o u n d d u r i n g r e g e n e r a t i o n ( C o u l o n et al., 1976). A f t e r cephalic a m p u t a t i o n , the regenerative p r o p e r t i e s o f Owenia decrease following an a n t e r o - p o s t e r i o r g r a d i e n t (Thouveny, 1967). A t the level o f the 1st a b d o m i n a l segment r e g e n e r a t i o n is always complete, while at the level o f the 4th a b d o m i n a l segment it has never been observed, a l t h o u g h a s t i m u l a t i o n o f resting cells takes place after t r a u m a ( C o u l o n a n d Marilley, u n p u b l i s h e d results). It has been suggested t h a t m o n o a m i n e n e u r o t r a n s m i t t e r s m i g h t p l a y an i m p o r t a n t role in regenerative processes in invertebrates ( C h a p r o n a n d C h a p r o n , 1972; F r a n q u i n e t et al., 1976; C o u l o n a n d Marilley, 1978). Thus, it was the aim o f the p r e s e n t s t u d y to detect the presence o f i n d o l a m i n e a n d c a t e c h o l a m i n e n e u r o n s at light a n d electron m i c r o s c o p i c levels, t a k i n g a d v a n t a g e o f the r e u p t a k e c a p a c i t y o f these neurons.
Materials and Methods
Autoradiography The 1st and 4th abdominal segments from specimens of Oweniafusiformis were incubated in sea water for 20 to 60min with: a) 3H-5-hydroxytryptamine (5-HT) (Amersham/Searle, specific activity 21 Ci/mM, concentration 7.10- SM); b) 3H-noradrenaline (NA) (CEA/Saclay, specific activity 20 Ci/mM, concentration 10-7M). In some cases the labeled transmitters were supplemented with unlabeled ones or with a drug: a) 3H-5-HT (10-TM) and cold NA (10-6M). b) 3H-5-HT (10-TM) and fluoxetin (2.10-6M). c) 3H-5-NA (10-7M) and cold 5-HT (10-6M). The radioactive tracers were preserved from chemical oxidation by addition of 0.002 700ascorbic acid. After incubation, all samples were washed for 15 min in sea water with the corresponding nonradioactive transmitter (10-4 M), then fixed in 2 ~ glutaraldehyde in cacodylate buffer and sea water. For light microscopic autoradiography, the pieces were embedded in paraffin. The sections (7 pin thick) were coated with Ilford K5 emulsion diluted 1 : 1 with distilled water, then exposed for 10 days. For electron microscopic autoradiography, the samples were postfixed in osmium and embedded in Araldite. The ultrathin sections were coated with Ilford L4 emulsion diluted 1 : 4 (Larra and Droz, 1970), exposed for 21 days, then developed in Kodak D 19b. The grids were observed with a HitachiHU 12 electron microscope.
Quantitative Uptake A study of quantitative uptake was performed according to Ternaux et al. (I 977). Portions of the nerve cord at different body levels of seven pooled animals were homogenized in buffered artificial sea water (in mM : Tris-Hcl pH 7.5,10; NaC1, 494; CaC12,10; K C1,10; MgSO4, 30; Mg C12,20) with ascorbic acid and sucrose, and transferred to Eppendorf microtubes containing 400 ~tlof the same medium. 3H-5-HT (7.10-SM) or aH-NA (7.10-aM) was added and incubation was performed for 8min at 25~ The reaction was stopped by adding 400~tl of non-radioactive sea water and incubating at 0~ After
Indolamine and Catecholamine Neurons of Oweniafusiformis
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centrifugation for 4min at 9,800 x g, the pellet was washed with 400~tl of artificial sea water, and suspended in 200 ~tlof water containing Triton X-100 (0.5 %) for scintillation counting. An aliquot of the homogenate was sampled for protein determination. Controls were obtained by incubating samples at 0~ Results In Owenia, the medio-ventral nerve cord is in an intra-epithelial position. The nerve fibers are located on the external side of the basal lamina, and the perikarya are scattered a m o n g epidermal cells. Axonal processes extending to the dorsal side are found laterally between the basal lamina and the epidermis (Fig. 1) (Coulon and Thouveny, 1974).
In vitro Incubation in 3H-5-HT The time course of labeling studied for periods varying from 20 to 60 min gives identical autoradiographic reactions. Following 3H-5-HT administration, in cross sections at the level of the 1st abdominal segment a diffuse autoradiographic reaction is observed on all structures; it is more pronounced on the epidermal cells. In contrast, dense and selective silver grain accumulations are located in the central part of the nerve cord and in its lateral (Figs. 1,2) and dorsal processes. In this respect the serial sections show a uniform pattern of the autoradiographic reaction. At the level of the 4th abdominal segment, there is a decrease in the selective reaction in the nerve cord and the axonal processes (Fig. 3). Addition of cold NA at a tenfold concentration produces the same autoradiographic pattern at both levels, i.e. the 1st (Fig. 4) and the 4th abdominal segments. On the other hand, after incubation in aH-5-HT and fluoxetin, a selective autoradiographic reaction on the nerve cord and lateral fibers is no longer found, although the diffuse reaction and a more pronounced one on the adjacent epidermal cells persist (Fig. 5). The labeled structures can be identified by means of electron microscopic autoradiography. Silver grain accumulations located in juxtaposition to the basal lamina at the level of the ventral nerve cord or at the level of the lateral and dorsal axonal processes correspond to axonal varicosities containing dense core vesicles (60-90 nm in diameter) (Fig. 9). In the central part o f the nerve cord, silver grains are seen mainly on the axonal fibers. At the periphery of the nerve cord, some perikarya show an autoradiographic reaction over the nucleus and the cytoplasm (Fig. 10). Study of the quantitative uptake by scintillation counting shows a decrease of the radioactivity in the nerve cord following an antero-posterior gradient. The maximal value is obtained at the level of the 1st segment, while minimal values are seen at the level of both the 3rd and 4th segments (Table 1).
In vitro Incubation in 3H-NA The autoradiographic reactions are similar after an incubation time varying from 20 to 60 min.
Figs. 1-3. Autoradiographs of cross sections of Owenia following incubation with aH-5-HT for 30 min (dark field illumination, Figs. 2 and 3). Fig. 1. Medio-ventral position of the nerve cord (nc) in epidermis (e) and the lateral axonal processes (ap). Note the intense labeling in the central part of the nerve cord; brn basal lamina (membrane); m muscle. • 300. Fig. 2. First abdominal segment. Three types of A R G reactions are observed (see text). Note the selective labeling in the central part of the nerve cord (nc) and along the lateral axonal processes (arrows). x 300. Fig. 3. Fourth abdominal segment. The intensity of the labeling decreases in the ventral nerve cord (nc). x 300
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Figs. 4 and 5. Dark field autoradiographs o f cross sections o f Owenia at the level o f the I st abdominal segment following 3H-5-HT incubation with cold NA (Fig.4) or fluoxetin (Fig.5). Fig.4. The distribution o f the labeling resembles that observed in Fig. 2. x 300. Fig. 5. Absence of silver grain accumulation over the nervous structures, the persistence o f a diffuse reaction and a more pronounced accumulation in the epidermal cells. • 300
Table 1. Quantitative fluctuations o f 3H-5-HT uptake and 3H-NA uptake in various areas of the nerve cord. Controls were obtained by incubating samples at 0~ (Mean values + standard error) Topography
Uptake of 3H-5-HT ( x 10-4 ~tCi/mg Prot.)
Uptake o f 3H-NA ( x 10-4 ~tCi/mg Prot.)
First abdominal segment Second abdominal segment Third abdominal segment Fourth abdominal segment Controls
34.17 + 26.3 + 17.5 + 17.0 + 6.9 +
6.79 ___0.48 5.80+ 0.61 4.69 ___0.22 5.23 + 0.73 1.4 + 0.09
3.82 5.86 0.71 1.0 0.21
Figs. 6-8. Dark field autoradiographs of cross sections of Owenia following 3H-NA administration alone (Figs. 6, 7) or with cold 5-HT (Fig. 8). Fig. 6. First abdominal segment. Silver grains are mainly located close to the basal lamina of the nerve cord. The axonal processes are poorly labeled, x 300. Fig. 7. Fourth abdominal segment. The distribution of the labeling resembles that observed in the 1st abdominal segment, x 300. Fig. 8. Fourth abdominal segment. Addition of cold 5-HT to the incubation medium does not alter the distribution of the label
Figs. 9and 10. Electron microscopic autoradiographs after incubation in 3H-5-HT for 30min. Fig. 9. Labeled varicosity containing numerous dense core vesicles (arrows) near an uniabeled one (*). Varicosities are surrounded by profiles of glial cells (gc). x 11,300. Fig. 10. Cell body showing positive reaction on nucleus and cytoplasm. • 20,000 Figs. 11 and 12. Electron microscopic autoradiographs after incubation in 3H-NA for 30min. Fig. 11. Uptake of aH-NA by axonal varicosities containing clear and dense core vesicles (arrows). • 16,500. Fig. 12. Labeled cell body. Note the presence of dense core vesicles, x 16,500
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At the level of the 1st abdominal segment, the 3H-NA reaction is less intense than the 3H-5-HT reaction. However, a selective labeling is seen in the central part of the nerve cord and near the basal lamina (Fig. 6). A reduced number of grains is usually observed in the lateral and dorsal fibers. The 4th abdominal segment exhibits principally the same distribution of the label (Fig. 7). In worms incubated with 3H-NA and tenfold concentrated cold 5-HT, a similar distribution of label is found at the level of both the 1st and 4th abdominal segments (Fig. 8). Ultrastructurally, labeled axonal varicosities contain vesicles with varying-sized dense cores (50-70 nm in diameter) (Fig. 11). Some cell bodies scattered among epidermal cells are also labeled (Fig. 12). Quantitative distribution of the radioactive material following 3H-NA incubation is comparable at the first two segments. Moreover, a slight decrease is found at the level of the 3rd and 4th segments (Table 1).
Discussion
Radioactivity observed in amine-containing tissues by autoradiography and by scintillation counting may be due to the radioactive amines and/or their metabolites. Monoamine oxidase is relatively inactive in invertebrates (Hiripi and Salanki, 1971), but a recent biochemical study has shown the formation of sugar conjugates after 1 to 15 h following in vitro administration of labeled serotonin in Aplysia (Goldman and Schwartz, 1977). Thus it is probable that after a shorter incubation time, the major part of radioactivity arises from the amine itself, which was protected from chemical oxidation by an antioxidant. Concerning autoradiographic (ARG) studies, three types of reactions should be regarded separately: 1) The diffuse, non-selective ARG reaction is usually observed after incubation with each amine. This type corresponds to an artefactual retention by glutaraldehyde of the free or weakly bound monoamines and their metabolites and indicates tracer penetration into the tissue (Descarries and Droz, 1970). 2) The more pronounced reaction, which can be seen on some structures of the epidermis mainly in the sagittal part of the first abdominal segment, might be attributed to a retention capacity of the epidermis, which varies following an antero-posterior gradient (Thouveny, unpublished results). 3) The intense and selective silver grain accumulation indicates the sites where the tracer has been taken up and retained. Published data obtained in vertebrates as well as in invertebrates indicate that the intensely labeled structures following 3H-5-HT and 3H-NA administration are monoaminergic neurons (Descarries and Dupin, 1974; Goh and Davey, 1976, Bessone et al., 1978). Furthermore, following in vitro incubation with the labeled transmitters at low concentrations (10-7M and below), autoradiography and analysis of quantitative uptake allow a specific detection of serotoninergic or catecholaminergic neurons in vertebrates (Shaskan and Snyder, 1970) as well as in invertebrates (Bessone et al., 1978). Based on this general knowledge, our results in Owenia suggest the existence of different kinds of monoaminergic neurons:
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1) Different autoradiographic patterns are observed following 3H-5-HT and 3H-NA incubation, respectively. 2) Addition of unlabeled NA or 5-HT to the incubation medium does not change the distribution of silver grains in tissues incubated previously with 3H-5-HT or 3H-NA. 3) The absence of an intense A R G reaction following addition of fluoxetin (a selective inhibitor of 5-HT uptake by serotoninergic neurons; H a m o n , 1975) appears to prove the existence of serotoninergic neurons. These results are confirmed by a study of the quantitative uptake and are in agreement with biochemical assays of 5-HT performed in another polychaete, Glycera (Manaranche, 1975). On the other hand, neurons labeled following 3H-NA administration may be defined as catecholaminergic (CA) since CA neurons have the ability to take up and retain nonspecifically noradrenaline or dopamine tracers (Descarries and Droz, 1970). Morever, they are probably noradrenergic, since, in comparison to noradrenaline, a very weak concentration of dopamine has been found by biochemical assays in another polychaete annelid (Manaranche, 1975). At the ultrastructural level, the structures intensely labeled following 3H-5-HT or 3H-NA administration correspond to those studied light microscopically, and therefore can be considered as serotoninergic or noradrenergic varicosities. The aspect of their granular vesicles appears to differ ultrastructurally; however, in other species, a uniform type of vesicle has been found in tissues storing different amines (Pellegrino de Iraldi et al., 1971). The analysis of the quantitative uptake indicates the predominance of 5-HT innervation in the ventral nerve cord of Owenia; this is in agreement with biochemical determinations of 5-HT in other invertebrates (Gerschenfeld, 1973; Manaranche, 1975). Moreover, the decrease in neurotransmitter uptake in the 4th abdominal segment might be related to the decrease in the density of the monoaminergic innervation and/or to a modified activity of these fibers. It has to be proven whether this peculiarity m a y be related to the loss of the regenerative capacity of the 4th abdominal segment.
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Accepted December 10, 1978
