ZOOLOGICAL SCIENCE 8: 905-913 (1991) 199] Zoological Societj ofJapan Distribution of Methionine-Enkephalin-Like and FMRFamide-Like Immunoreactivities in the Central Nervous System (Including Dorsal Bodies) of the Snail Helix aspersa Muller Claude Roland Marchand, Bernadettf Griffond, Khalid Mounzih and Claude Coi ard Laboratoire de Zoologie el Embryologie, SDl ( \R\ 6319, Faculti des Sciences, place Leclerc, 25030 Besancon Cedex, France — ABSTRACT We investigated the immunocyto-localization oi methonine-enkephalin and of FMRFa- mide in the central nervous system and in the dorsal bodies o\ adult snails Helix aspersa. The results summarized on maps demonstrate that: 1) Methionine-enkephalin- and FMRFamide-like substances are detected in many neurons of all ganglia. They are never colocalized. 2) The areas of supra- and subesophageal methionine-enkephalin positive dorsal bodies are innervated b\ a network of FMRFamide positive fibers, which contain immunogold positive granules and establish close contacts with the dorsal-body cells. Theextensivedistributionofmethionine-enkephalin-and FMRFamidefibersandcellsofvarioussizes suggeststhatthesepeptidesplaydifferent functionsin the nervoussystem. Aspecial attention ispaid to the control of the dorsal bodies according to previous functional results. organs [5-8], but the hormones of the dorsal INTRODUCTION bodies have not yet been identified. They are In Helix aspersa, as in other Stylornmatophora, under inhibitory nervous control, as demonstrated the central nervous system (CNS) is built up of 9 by in vivo and in vitro experiments [6, <S-1()|. and ganglia surrounded by a connective tissue sheath, their innervation probably originates from the the perineurium, which contains non-nervous en- Cerebral Green Cells (CeGC). docrine cells, the so-called dorsal bodies. There have been some papers reporting the The neurosecretory cells of the circumesopha- existence of methionine-enkephalin [11] and geal ganglia have been studied by classical FMRFamide (12. 13] immunoreactivities m Helix methods (chrome-haematoxyiin, paraldehyde- CNS. The present papercompares the distribution fuchsin) [1] and by the alcian blue-alcian yellow of substances related to these both peptides in the technic [2. 3]. The use of immunocytochemistry CNS of Helix aspersa. It points out the abundance has recently given complementary information and of neurons with immunoreactivity like that ol mav help to define neuronal sub-populations. either peptide, the abundance ofaxons innervating Moreo\er the supra- and sub-esophageal localiza- the cloisal bod) areas and their immunoreactivity tion of the dorsal body cells has been made more mainly to anti-FMRFamide. precise b\ their immunoreactivit\ with antibodies to methionine-enkephalin [4]. However it is well MATERIALS AND METHODS established that the dorsal bodies of Helix aspersa are endocrine organs which control vitcllogencsis Animals and the synthetic activity of the female accessor) Adult snails were bred undercontrolled arhfu lal Accetpted June 20. 1991 conditions(long-da) lightcycles 18hrL-6hi D ii Received Ma> s. 1991 20 ( and 9Q9t relative humidity) and fed ad iibi- 906 C. R. Marchand, B. Griffond, K. Mounzih and C. Colard turn in the Centre Universitaire d'Heliciculture de karya and fibers in the different ganglia, in the Besangon. neuropiles and in the different commissures and All of the animals used in these investigations connectives. (10) were sexually active and were sacrificed just before mating. Methionine-enkephalin immunoreactivity: Immunoreactive cells of different sizes were Methods observed in all ganglia (Figs. 1,2). In the cerebral The circumesophageal nerve rings were excised ganglia, they are small. Most ofthem are located and immediately immersed in Bouin-Hollande on the dorsal surface. A few cells of 10jum in fixative (+10% HgCl2) for 24hours. The 6jum diameter are scattered in the procerebrum, espe- paraffin sections were processed for immunocy- cially in the medial and latero-dorsal regions. In tochemistry, as described previously, by the three the metacerebrum, 25-30jum positive cells are step method [4, 14-16] involving the peroxidase- dispersed dorsally or located along the lateral anti-peroxidase method [17]. Pieces ofconnective margins of the ganglia. Smaller cells (15-20/um) tissue containing dorsal body cells were removed extend along these margins or are concentrated at dorso-frontallytocerebralganglia, fixedfor2hrin the posterior region of the meta- and mesocereb- a mixture of 2% glutaraldehyde, 0.1 M sodium rum. As forthe so-calledCeGC, mostofthem are M cacodylate,0.17 NaCl (lv/lv/lv), washedover- methionine-enkephalin positive (Fig. 5), except M night in 0.1 buffer and embedded in epon- forsome whichform thelobesonthe anteriorpart araldite. Theultrathinsectionsweretreatedbythe of mesocerebrum. immunoglod method as reported earlier [18]. In the subesophageal ganglia, immunoreactive The primary antibodies were raisedin rabbits to cells are interspersed with negative cells ofsimilar the carboxy terminals of synthetic methionine- size. Concerning the immunoreactive cells, the enkephalin and FMRFamide and were used in pleural ganglia exhibit cells of 10 to 15jum in final dilutions of respectively 1/3000 and 1/8000. diameteratthefronto-dorsalregion and25-30jum These antibodieswere prepared andmaintainedin cells in a more posterior position. The parietal ourlaboratory [14, 19]. The two antibodiesdonot ganglia contain big cells, one or two cells of90-95 cross-react and are specific to the antigen they jum and cells of 50 to 60jum at the anterior part, were directed to [20]. In any case, the structural cells of40jum posteriorely. The visceral ganglion similarities between the two peptides led us to (Fig. 7) containscellsofvarioussizesamongwhich compare adjacent sections in order to determine some big positive cells (up to 100jum in diameter) whether peptides related to, or similar to, located dorsally and ventrally. In the pedal gang- methionine-enkephalin and FMRFamide are col- lia, a big cell (70jum) occurs on each side of the ocalized. midline onthe anteriorventralpartandmanycells Two maps of the intra- and extra-ganglionary of40-50jum form groups especiallyon the ventral localization of immunoreactive structures were surface. drawn by projecting each of them on to the same sketch. Particular attention was given to the FMRFamide immunoreactivity: Up to 250 CeGC [3] in order to establish the origin of the immunoreactive perikarya were counted in the nervous fibers innervating different areas in the ganglia and many fibers were seen in the connec- vicinity of the ganglia. tives and commissures (Figs. 3,4). In the cerebral ganglia, cells of various sizes are abundant in the meta-andmesocerebrum. Ineachmesocerebrum, RESULTS a big immunoreactive cell (80jum) is located just posterior to the inner part ofthe cerebral lobe. In Intra-ganglionic immunoreactive structures the CeGC area, many cells ofmedium size (40-50 Observations ofserial sections ofentire nervous jum) stain positively with the anti-FMRFamide systems have shown a lot ofimmunoreactive peri- serum. They send axons in different directions, Mk- and FMRF-like Substances in the Brain W7 MK Ventral map : \TX ;0 5*0) I/) FMRFa Dorsal map FMRFa Ventral map : I -i Mapsofdorsal (Figs. 1,3) and ventral (Figs. 2. 4) halvesofcentral nervoussystemsoi Helix aspersa The perikarya (irregularcircles)and fibers(dotsandshort lines) imfflunoreactive toaim methionine enkephalii 1. 2) or anti-FMRFamide (Figs. 3, 4) are shown ( cerebral ganglia; ( eG< cerebral green cells; DBa dorsal bodies area; P: pedal ganglia; PA: parietal ganglia; PI pkurai ganglia, \ viscera] ganglion 908 C. R. Marchand, B. Griffond, K. Mounzih and C. Colard t> Sfc N Cg'GC CeGC **v~ fc m*m&* 6 ^w^ <p I I % 8 0- £$ »*, ' #' ' T*wfjftpa> 10 : MR- and FMRF-like Substances in the Brain s>(W but particularly through the intercerebral commis- DISCISSION sure (Fig. 12). The biggest perikarya (100-120 ,um) are located ventrally in the visceral and This study shows the important distribution of parietal ganglia (Fig. 4). Whatever the conditions, two substances immunologically detected with there are little variations of the numbers of antibodies to. respectively, methionine-enkephalin FMRFamide positive cells in all ganglia and espe- and FMRFamide in perikarya and libers of the cially in the two mesocerebral lobes which never central nervous system of Helix aspersa. It contain FMRFamide positive perikarya. Adjacent confirms previous observations made on different sections treated either with anti-methionine- organs (aorta, gonad, nervous ring) |14, 15, 19, enkephalin orwith anti-FMRFamide show that the 21]. It also shows that methionine-enkephalin- and two peptides are never colocalized (Figs. 5, 6, 7. 8, FMRFamide-like substances are never colocal- 9, 10). ized, either in the neurons, or in the dorsal bodies. At the present time, all the animals observed in Extra-ganglionic immunoreactive structures anj physiological situation (young, adult, before Methionine-enkephalin immunoreactixit\ or after mating) never colocah/c methionine- The intra-connective endocrine cells (dorsal enkephalin and FMRFamide in their neurons. bodies) scattered in the vicinity ofthe anteriorpart In comparison with histochemical data, im- of the supra- and subesophageal ganglia specifical- munocytochemistry gives additional information: 1\ bind the anti-methionine-enkephalin (Fig. 9). e.g.. some unstained cells in the pedal ganglia [3] Sometimes, a large positive fiber is seen among are immunoreactive and are probably neurosecre- them in the supra-esophageal area (Fig. 11). We tory cells. Concerning the CeGC, the distribution have never observed methionine-enkephalin im- of immunoreactive cells partially overlaps the dis- munoreactive fibers in the subesophageal area. tribution ofalcian blue-alcian yellow positive cells. Immunocytochemistry however demonstrates the FMRFamide immunoreactivity: Numerous heterogeneity of the CeGC groups as some cells small immunoreactive fibers are visible among, are methionine-enkephalin positive, other are and often in contact with, the dorsal body cells in FMRFamide immunoreactive and other arc nega- the supra- (Figs. 10. 13. 14) as well as in the tive toward both antibodies: moreover the occurr- subesophageal (Figs. 15, 16) perineurium of the ence ofinsulin-related substances in these cells has central ner\ous system. They form a wide spread been recently reported [22, 23). This is worth network in the dorsal body area. The use of pursuing in depth (forexample by electron micros- immunogold technique confirms at the ultras- copy), so that an exact typology can be made. tructural level the presence of FMRFamide-like The large number of immunoreactive libers and substances in many nerves of the dorsal body area perikarya ol various sizes in all ganglia probably (Fig. 17) and in contact zones (synapse-like struc- reflects the diversity of functions of methionine- tures) between these nerves and the dorsal body enkephalin- and FMRFamide-like substances in cells (Fig. IS). Helix. In molluscs. FMRFamide and related subst- ances are known to pla\ physiological roles in various tissues (cardiac -iiul non cardiac muscles, nervous s\stem. glands) and to act as neuiotians Figs. 5-10. Immunocytochcmical detection of methionine-enkephalin (1 iga 5, 7. 9) and I MRFamidc like material I gs n. 8, 10) in the supra-esophageal if ius 5, 6), the subesophageal ganglia (Figs. 7. 8) and the dorsal bodies arealocatednearthecerebralganglia(Figs ('. I'M (Thearrowsshow thesameareaonadjacentsections) Fig! 5. 6. Adjacent sections to compare the localization of the immunoreacti\it\ m tin ( i < >< p. 7. X. Adjacent sections of the visceral ganglion - 460 Neithei the cerebral ganglia nor the visceral ganglion contain cells in which immunochemical Staining for the two peptides like is oolocalized (\ il-s 5, 6 and 7,8) I ig 9 Ihe dorsal bodycells are methionine-enkephalin immunoreactive - 460 f ig I" The dors.il bod) cellsdo not M ,ui with the antibod\ raised against FMRFamide but the) are innervated in FMRFamide positive fibers 910 C. R. Marchand, B. Griffond, K. Mounzih and C. Colard -- • "\fr'"v- ; i i ' I DBa :- DBa ' •'.*> *• . X i /- CeGC 12 CeGC DBa ^ v..,- 14 13 « DBa • VG 16 DBa DB 15 SLS . 17 18 :'.'/. Nik- and FMRF-like Substances in the Brai 911 mitters-neuromodulators in the central nervous logical to think that the majority of the FMRFa- system, as neuromuscular transmitters or as mide positive fibers innervating the subesophageal neurohormones [see 24-26]. Methionine-enke- dorsal bodies originate from the subesophageal phalin, on the other hand, is involved in several ganglia as suggested by immunocytochemistrv. processes such as locomotion, thermoregulation. Finally, the nervous control of dorsal body cells pain resistance and immunomodulation [see IS]. is probably more complex than expected, particu- Whereas the dorsal body cells of Limax max- larly since we have revealed methionine- imus are immunoreactive to antisera exhibiting enkephalin positive libers in the dorsal bodies in specificity for the C-terrainaJ end of FMRFamide addition to FMRFamide positive ones; more re- [27]. those of Helix aspersa do not give positive cently Van Minnen and Schallig [22] have de- reaction with our antiserum also raised against the scribed MIP-(Molluscan Insulin Peptide) im- C-terminal of FMRFamide. This divergent results munoreactive fibers in close association with the might be due to the use of different polyclonal dorsal body cells o\ Helix. antisera or to specific differences in the secretion In any case, it is difficult to follow the pathway products of the dorsal body cells. The dorsal of fibers from their origin to their end on serial bodies of Helix give a very strong reaction with an sections treated by the immunocytochemical tech- antibody to methionine-enkephalin. Moreover, an nics. Intraneuronal injections of lucifer yellow or important network of FMRFamide positive fibers other fluorescent substances will be helpful to is revealed, by immunocytochemistry. in the sup- precise where the dorsal bodies innervation comes ra- as well as in the subesophageal dorsal body from. area. The supra-esophageal dorsal bodies seem to be controlled in part by FMRFamide-like subst- ances, since synthetic FMRFamide and ACKNOWLEDGMENTS pQDPFLRFamide inhibit their in vitro protein The authors thank B. Jolibois for typing the manu- synthesis [20. 28] and since FMRFamide-like sub- script and B. Regent for printing the photographs. stances are present in granules ofmany nerves and synapse-like structures of the dorsal body area REFERENCES [29]. 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