Table Of ContentZOOLOGICAL 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
:
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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
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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 '
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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]. The CeGC might exert this inhibitorycontrol
[10] and the present results are consistent with this Kuhlmann. I). ( 1963) Neurosecretion bei Heliciden
notion because many CeGC are FMRFamide posi- (Gastropoda). Z. Zellforsch., 60: 909
ti\e. However, our results further do not exclude Kai-Kai. M. A. and Kerkut. G. A. (1979) Mapping
the possibility that FMRFamide positive cells from abrnadinulotfraHsetlriuxctuarseperosla.neCuormopse.crBeitoolryhemcellsPhyisniolt.h.e
other neuronal groups are also involved in the 64A: 97-107.
innervation of this area. In the same wav it is Wijdenes, J., Van Minnen, J. and Boer. H. H.
Fig 11 Immunocytochemical detection ofmethionine-enkephalin-like material in the supra-esophageal part ofthe
nerve ring. The dorsal body area (DBa) isstrongly methionine-enkephalin positive, as is a nervous fiber (arrow)
traversing the area, probablv coming from the cerebral ganglia, where some immunoreactive CeGC are visible.
- 125.
12-16. Immunocytochemical detection ol FMRFamide-like material in the SUpra-esophageal part ol the nerve
ring (Figs. 12. 13. 14) and near the visceral ganglion (Figs 15. 16). Fig. 12 ( • 290) and 13 ( - 460). Path ol
FMRFamide positive fibers; among the tntra-commissural positive fibers(large arrow >. one isdirected toward the
DBa (small arrow). Fig. 14. Small CeGC ol the right lobe and intra DBa fibers (arrow) are FMRFamide
positive -290 Fig. 15. ThesubesophagealDBaisinnervatedbyFMRFamidergicaxons(blackpoints). <460
Fig. 16. One might suppose that the FMRFamidergic fibers innervating the subesophageal DBa (arrow ) originate
from the visceral ganglia (VG) • 290.
Fig. 17. Anti-FMRFamide positive axon m the dorsal bodv area Gold particles are concentrated on the granulesol
the axon X31000.
Fig. 18. Synapse-like structure (SLS) between a small nerve and a dorsal bod) cell (DB) The gold particlesare very
numerous on the granules of the SIS whereas the cytoplasm oi the DB cell is not Libelled • 29300
:
912 C. R. Marchand, B. Griffond, K. Mounzih and C. Colard
(1980) A comparative study on neuro-secretion 15 Marchand, C. R. andDubois, M. P. (1985) Miseen
demonstrated by the Alcian blue-Alcian yellow evidence parimmunocytologie de sitesantigeniques
technique in three terrestrial Pulmonates (Stylom- fixant des anticorps anti-aMSH et anti-methionine-
matophora). Cell Tiss. Res., 210: 47-56. enkephaline dans l'ovotestis de l'escargot adulte
4 Marchand, C. R. and Dubois, M. P. (1986) Im- Petit-GrisHelixaspersa. C. R. Acad. Sci. Paris,301:
munocytochemical and ultrastructural evidence for 233-238.
supra- and subesophageal localization of dorsal- 16 Marchand, C. R. and Dubois, M. P. (1986) Im-
body cells of the snail Helix aspersa. Gen. Comp. munoreactivity of the hermaphroditic gland of the
Endocrinol., 63: 374-380. snail Helixaspersa Miillertowardsantibodiesraised
5 Wijdenes,J.,Vincent, C, Griffond, B. andGomot, to fragments of pre-pro-opio-melanocortin. Cell
L. (1983) Ultrastructural evidence forthe neuroen- Tiss. Res., 245: 337-341.
docrine innervation of the dorsal bodies and their 17 Sternberger, L. A., Hardy, P. H., Cuculis,J. J. and
probablyphysiologicalsignificanceinthepulmonate Meyer, H. G. (1970) The unlabeled antibody en-
snail Helix aspersa. In "Molluscan neuro- zyme method of immunohistochemistry: Prepara-
endocrinology". Ed. by J. Lever and H. H. Boer, tionandpropertiesofsolubleantigen-antibodycom-
North-Holland Publ., Amsterdam/Oxford/New plex (horseradishperoxidase-antihorseradishperox-
York, pp. 147-152. idase) and its use in identification ofspirochetes. J.
6 Vincent, C., Griffond, B., Gomot, L. and Bride, J. Histochem. Cytochem., 18: 315-333.
(1984) Etude in vitro de l'influence des corps 18 Magdelaine, S., Marchand, C. R. and Griffond, B.
dorsaux sur 1'ovogenese d'Helix aspersa Miiller. (1990) Electron microscopic study of the optic
Gen. Comp. Endocrinol., 54: 230-235. tentacle collar cells of the snail Helix aspersa and
7 Griffond, B. and Vincent, C. (1985) Etude de immunolocalization of methionine enkephalin-like
l'activitedescorpsdorsauxdel'escargotHelixasper- material in their granular content. Biol. Cell., 68:
sa Miiller au cours des phases physiologiques de la 245-250.
vie adulte etsousdifferentesphotoperiodes. Intern. 19 Marchand, C. R., Wijdenes, J. and Schot, L. P. C.
J. Invert. Reprod. Dev., 8: 27-37. (1982) Localisation par la technique cyto-immuno-
8 Bride, J. and Gomot, L. (1988) La synthese poly- enzymologique d'un neuropeptide cardio-excitateur
saccharidique in vitro de l'oviducte d'Helixaspersa (le FMRF-amide) dans le collier peri-oesophagien
un bioessai pour eprouver l'activite d'organes en- $HelixaspersaMiiller(Gasteropode,Pulmone,Sty-
docrines, la gonade et le complexe neuro-endocrine lommatophore). C. R. Acad. Sci. Paris,294: 39-44.
cephalique. C. R. Acad. Sci. Paris, 307: 199-204. 20 Mounzih, K. and Griffond, B. (1988) Effet d'un
9 Vincent,C, Griffond,B.,Wijdenes,J. andGomot, heptapeptide apparente au FMRF-amide (pQDPF-
L. (1984) Controle d'une glande endocrine: les LRFamide) sur l'incorporation d'acides amines par
corps dorsaux par le systeme nerveux central chez lescorpsdorsaux&Helixaspersainvitro. C. R. Soc.
Helixaspersa. C. R. Acad. Sci. Paris,299: 421-426. Biol., 182: 572-578.
10 Wijdenes, J., Schluter, N. C. M., Gomot, L. and 21 Griffond, B., Boer, H. H. and Wijdenes, J. (1986)
Boer, H. H. (1987) In the snail Helix aspersa the Localization and function of an FMRF-amide-like
gonadotropic hormone-producing dorsal bodies are substance in the aorta of Helix aspersa. Cell Tiss.
under inhibitory nervous control ofputative growth Res., 246: 303-307.
hormone-producing neuroendocrine cells. Gen. 22 Van Minnen, J. and Schallig, H. D. F. H. (1990)
Comp. Endocrinol., 68: 224-229. Demonstration of insulin-related substances in the
11 Ortiz, T., Pinero, J. and Covenas, R. (1987) central nervous systems ofpulmonates and Aplysia
Metenkephalin-like immunoreactivity in the ner- californica. Cell Tiss. Res., 260: 361-386.
voussystemofHelixaspersa. Zool. Sci.,4: 743-746. 23 Gomot, A., Gomot, L., Marchand, C. R., Colard,
12 Lehman, H. K. and Price, D. A. (1987) Localiza- C. and Bride, J. (1991) Immunocytochemical loca-
tion of FMRFamide-like peptides in the snail Helix lization of insulin-related peptide(s) in the central
aspersa. J. exp. Biol., 131: 37-53. nervous system of the snail Helix aspersa Miiller.
13 Elekes, K. andNassel, D. R. (1990) Distributionof Involvementingrowthcontrol. Cell. Molec. Neuro-
FMRFamide-like immunoreactive neurons in the biol., in press.
central nervous system of the snail Helix pomatia. 24 Raffa, R. B. (1988) The action of FMRFamide
Cell Tiss. Res., 262: 177-190. (Phe-Met-Arg-Phe-NH2) and related peptides on
14 Marchand, C. R. and Dubois, M. P. (1982) Detec- Mammals. Peptides, 9: 915-922.
tion immunocytologique de materiel apparente a 25 Cottrell, G. A. (1989) The biology ofthe FMRFa-
differents peptides de Vertebres dans le collier ner- mide-series of peptides in molluscs with special
veuxdel'escargot(HelixaspersaMiiller).J. Physiol. reference to Helix. Comp. Biochem. Physiol., 93A:
Paris, 78: 595-598. 41-45.
Mk- and FMRF-like Substances in the Brain 913
26 Kobayashi. M. and Muneoka. Y. (1989) Functions. inhibition of methionine incorporation in the dorsal
receptors, and mechanisms of the FMR1 amide- bodies of Helix aspersa b\ synthetic FMRFamide.
related peptides. Biol. Bull.. 177: 206-209. Comp. Biochem. Physiol., MC: 45-49.
27 Cooke. I. R. C. and Gelperin, A. (1988) Distribu- 2(> Griffond, B. andMounzih, K. (1990) Innervationof
tion o\ FMRFamide-like immunorcactivitv in the the dorsal bod\ cells of Helix aspersa. immunoc\-
nervous system o\ the slug Limax maxunus. Cell tochemical evidence tor the presence of FMR] .i
Tiss. Res.. 253: 69-76. midc-likc substances in nerves and synapses. Tiss.
28 Griffond, B. and Mounzih. K. (1989) /,; vitro Cell. 22: 741-748.
