Download pdf - Neurons in a variety of molluscs react to antibodies raised against the VD1/RPD2 ?-neuropeptide of the pond snail Lymnaea stagnalis

Celt Tissue Res (1993) 273:371-379 Cell Tissue

Research �9 Springer-Verlag 1993

Neurons in a variety of molluscs react to antibodies raised against the VD1/RPD 2 e-neuropeptide of the pond snail Lymnaea stagnMis R.M. Kerkhoven 1, M.D. Ramkema ~, J. Van Miunen 1, R.P. C roll2, T. Pin 3, H.H. Boer 1

1 Department of Organismic Zoology, Faculty of Biology, Free University, De Boelenlaan 1087, NL-1081 HV Amsterdam, The Netherlands 2 Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3R14H7, Canada 3 Laboratoire de Neurobiologie, C.N.R.S., 31 Chemin Joseph-Aiguier, F-13402 Marseille Cedex 9, France

Received: 26 October 1992 / Accepted: 27 January 1993

Abstract. The VD 1 and RPD2 neurons of Lyrnnaea stag- halls innervate other central neurons, certain skin areas, the pneumostome area, and the auricle of the heart. Re- cently, a set of four (5, e, e, fl) neuropeptides produced by these giant neurons and by certain other central neurons has been characterized. Although alternative splicing of the preprohormone of these neurons yields at least 10 different e neuropeptides, an affinity-purified antiserum directed against a domain common to all a neuropeptides has previously been shown to be highly selective in staining VDI, RPD 2 and other neurons that produce the preprohormone. Since the gene encoding the neuropeptides is structurally similar to that expressed in R15 of the marine opisthobranch Aplysia californica, we have used the affinity purified antiserum as a marker for V D J RPD2-related systems in other molluscs. Immunoposi- tive neurons and fibers are observed in the central nervous systems of all species studied (Achatina fulica, An- odonta sp., Aplysia brasiliana, A. californica, Bulinus truncatus, Cepea sp., Eobania vermiCulata, Helix aspersa, H. pomatia, Limax maximus, Mytilus edulis, Nassarius reticulatus, V iviparus viviparus). Several medium-sized, and small neurons and 1-4 giant neurons are found in the pulmonates and opisthobranchs. The giant neurons in pulmonates have locations in the subesophageal ganglion, axonal branching patterns, and terminal arboriza- tions in the auricle of the heart; all these characteristics are similar to those of VD 1 and RPD 2. Double-labellihg (Lucifer yellow injection, immunocytochemistry) con- firms that the two giant neurons in Helix pomatia are Br and Br'. The immunoreactive ceils in A. fulica appear to include the VIN and PON neurons. The antiserum also stains cells that appear to be the R15 neurons in two Aplysia species. The small and medium-sized neurons are distributed widely over the central ganglia of opisthobranchs and pulmonates. In prosobranchs and bivalves, small neurons are found in the cerebral and abdominal ganglia. No evidence has been found for innervation of the heart in these latter g roups but in M. edutis, im-

Correspondence to: R.M. Kerkhoven

munoreactive terminals can be observed in the gill. The results suggest the evolutionary conservation of immunoreactive peptides and the neurons that produce them, and thus support and extend previous hypotheses regarding the homology of certain giant neurons across molluscan species.

Key words: Cardioactive peptide - Immunocytochem- istry - Bivalve - Gastropod - Pulmonate - Opistho- branch - Lymnaea stagnalis (Mollusca)

Introduction

VDt and RPD2 are giant neurons in the visceral and right parietal ganglion, respectively, of the pond snail Lymnaea stagnalis (Soffe and Benjamin 1980). The neurons are electrotonically coupled and generate a bursting pacemaker potential with a frequency of approximately 1 Hz (Van der Wilt et al. 1987). ReCently, a neuropeptide gene exPressed by VD1 and RPD2 has been cloned and Characterized:(B0gerd et al. 1991). Four neuropeptide domains (5, ~, e and fl) are present on the VD~/RPD2 preprohormone, but more than 4 peptides are produced by the cells, since as many as 10 different forms of the a neuropeptide may be synthesized because of alternative splicing of the m RN A (Bogerd et al. 1993). In situ hybridization experiments ha;ce shown that VD1/RPDz gene expression is not restricted to the two giant neurons, but also occurs in approximately 70 medium-sized and small neurons located in the cerebral, the visceral, the right parietal and the pedal ganglia (Kerkhoven et al. 1992). These neurons can be divided into three classes on the basis of their different reactions to VD1/RPD2- specific antisera, indicating that they have a variable peptidergic content (Kerkhoven et al. 1992).

VD~ and RPD2 react very strongly with an affinity purified antiserum directed against a peptide domain common to all e neuropeptides (the C-terminal domain of 9 amino acids: H H P R N C G F N ) . This el-antiserum

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has been used in c o m b i n a t i o n wi th t racer dye studies to ident i fy the targets o f VD1 and RPD2 ( K e r k h o v e n et al. 1991). A x o n s o f these neu rons have been obse rved to b r anch extensively in the cent ra l ne rvous system (CNS) (see also Soffe and Benjamin 1980). F u r t h e r m o r e , pe r iphe ra l p ro jec t ions have been no ted in cer ta in skin areas, the p n e u m o s t o m e area , and t h e auricle o f the hea r t ( K e r k h o v e n et al. 1991). These m o r p h o l o g i c a l ob- serva t ions are s u p p o r t e d by exper imen t s ind ica t ing tha t cer ta in fo rms o f the c~ n e u r o p e p t i d e enhance the bea t f requency and a m p l i t u d e o f the auricle o f the hea r t (Bo- gerd 1992), and tha t the cells m a y also be involved in r e sp i r a to ry func t ions (Van der Wi l t et al. 1987).

Several lines o f evidence suggest t ha t o the r mol luscs m a y con ta in h o m o l o g s o f the V D I / R P D 2 system. Fi rs t , the V D 1 / R P D 2 p r e p r o h o r m o n e encoded by the gene appears to be s imi lar in a m i n o acid s t ruc ture to a p r e p r o - h o r m o n e o f the g ian t n e u r o n R15 o f the d i s t an t ly re la ted ( M o o r e and P i t r a t 1960) mar ine o p i s t h o b r a n c h Aplysia californica (Buck et al. 1987; Bogerd et al. 1991). Sec- ondly , several o the r g a s t r o p o d s possess neurons tha t share s imi lar bu r s t ing ac t iv i ty pa t t e rn s and s imi lar loca- t ions to R15 and V D 1 / R P D 2 . Such cells inc lude cell 11 in Otala lactea (Ga ine r 1972; I fshin et al. 1975), V I N and P O N neurons in Achatinafulica ( G o t o et al. 1986; F u r a k a w a and K o b a y a s h i 1987; see also Chase and G o o d m a n 1977), Br and Br' neu rons in Helix aspersa (Kerku t et al. 1975; Pin and G o l a 1983), and the H Z cell in Clione limacina (Ar shavsky et al. 1990). Phys io- logical and m o r p h o l o g i c a l evidence has p rev ious ly been o b t a i n e d showing tha t these g ian t neurons are involved in the r egu la t ion o f hea r t func t ions (Ifshin et al. 1975; Pin and G o l a 1983; F u r a k a w a and K o b a y a s h i 1987; Ar - shavsky et al. 1990).

Figs. 1-4. Immunocytochemical staining, with the el-antiserum, of neurons and fibers in sections of the central nervous system of some pulmonates

Fig. 1 A-E. Bulinus truncatus. A Giant neuron (arrow) in the visceral ganglion, x 110. B Giant neuron (arrow) in the right parietal ganglion. • 110. C Medium-sized neuron (arrow) near the lateral lobe in the cerebral ganglion, x 115. D, E Small neurons in the cerebral ganglion, (D, arrow) and pedal ganglion (E, arrow). D x 220; E x 205

Fig. 2A-F. Achatina fulica. A-D Suboesophageal ganglion, A, B Location of the giant neurons in the ganglion (arrows). x 50. C Higher magnification of a giant neuron (large arrow) with its thick initial axon (small arrow) that branches in the neuropil (asterisk). x 200. D Medium-sized neurons (arrows). x 150. E, F Axonal terminals (arrows) among neurons (asterisks) in the pedal ganglia. x 200

Fig. 3A-D. Limax maximus, subesophageal ganglion. A Morpholo- gy of the branching (small arrows) of the initial axon of one of the giant neurons (large arrow), x 130. B--D Locations of the three giant neurons (solid arrows) in the ganglion; open arrows moderately stained medium-sized neurons, x 50

Fig. 4A, B. Cepea sp. A Intensely (large arrow) and moderately (small arrows) stained giant neurons in the subesophageal ganglia. x 85. B Note the branching (small arrow) of one of the intensely stained giant neurons (large arrow), x 85

The p resen t c o m p a r a t i v e s tudy was u n d e r t a k e n us ing the e l - a n t i s e r u m as a m a r k e r for cells tha t migh t be h o m o l o g o u s to the V D 1 / R P D 2 sys tem o f L. stagnalis. The m o r p h o l o g y o f i m m u n o r e a c t i v e neurons pe rmi t s a fur ther eva lua t ion o f homolog ie s f rom across a b r o a d spec t rum o f mol luscs .

Materials and methods

Animals

Representatives of maj or molluscan groups were studied. Pulmona- ta: Bulinus truneatus (Basommatophora); Aehatina fulica, Limax maximus, Cepea sp. Eobania vermiculata, Helix aspersa, H. pomatia (Stylommatophora); Opisthobranchia: Aplysia californica and A. brasiliana (kindly provided by Dr. A. ter Maat); Prosobranchia: Viviparus viviparus (Meso-Gastropoda), Nassarius reticulatus (Neo- Gastropoda); Bivalvia: Mytilus edulis and Anodonta sp. B. truncatus were bred in the laboratory under controlled conditions (12 h light-12 h dark, 25 ~ C water temperature). The other species were collected in the field.

Preparation and fixation of tissues

After removing the shell (if necessary), the CNS, the heart, and occasionally other organs were dissected from each specimen and fixed in Bouin's mixture for 3 days at 4 ~ C. The tissues were dehydrated through an alcohol series and then embedded in paraffin (melting point 54 ~ C). Sections of 7 gm thickness were made (cf. Kerkhoven et al. 1990).

Immunocytochemistry

Immunocytochemical staining was performed according to the two- step method of Sternberger (1986). To remove endogenous peroxi- dases, tissue sections were incubated for 45 rain in methanol containing 1% acetic acid and 0.1% H202. Rehydrated sections were washed with incubation buffer (TRIS-Tween): 0:1 M TRIS/HC1, pH 7.4 containing 0.05% Tween-20. Sections were incubated over- night (4 ~ C) with cd-antiserum (diluted 1 : 500 in TRIS-Tween). The cd-antiserum is a polyclonal rabbit antiserum raised against syn- thetic cd-neuropeptide (21 amino acids: DMYEGLAGRCQH- HPRNCPGFN) as predicted from the VD1/RPD2 neuropeptide cDNA (Bogerd et al. 1991; Kerkhoven et al. 1991). The cd-antiserum was affinity-purified using the C-terminal peptide domain (see Introduction) common to all ~-neuropeptides (Bogerd et al. 1993). Unpurified antiserum was used for immunocytochemical staining of the CNS of A. brasiliana and the heart of A. californiea. The sections were subsequently washed twice with TRIS-Tween and incubated (1 h at 20 ~ C) in swine anti-rabbit antibody conjugated to peroxidase (Dako Immunocytochemical Products, Denmark) diluted 1 : 100 in TRIS-Tween. The sections were washed with TRIS- Tween and subsequently in TRIS buffer without Tween-20. The peroxidase reaction was developed in a chromogen mixture containing 0.05% 3,3'-diaminobenzidine-4HC1 (Sigma, St. Louis, Mo.) and 0.01% H202, for 5 10 min at 20 ~ C. Positive controls consisted of treating sections of the CNS of L. stagnalis with the cd-antiserum (see Kerkhoven et al. 1992). The sections were dehydrated in an ascending ethanol series, cleared in xylene and mounted in Entallan (Merck).

Lucifer yellow filling of Br and Br' of Helix pomatia

Helix pomatia were decapitated and the CNS was dissected. The ganglia were then placed in saline (57 mM NaC1, 5 mM KC1, 8 mM

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CaC12, 8 mM MgCl2 and 10 mM HEPES, adjusted to pH 7.4) and the connective tissue sheath was partially removed from the vicinity of Br and Br'. These cells were identified by visual inspection and by physiological properties revealed upon microelectrode penetra- tion. Lucifer yellow (LY, 10% in saline, Sigma) was then injected iontophoretically into these neurons for 2 h (20-40 nA pulses of 500 ms duration and at a frequency of 1 Hz; cf. Pin and Gola 1984a, b). The semi-intact preparations were subsequently fixed for 24 h at 4 ~ C in 4% paraformaldehyde, dehydrated and embedded in paraffin. In rehydrated sections (7 gm thick) of the CNS, the LY fluorescence of Br or Br' was photographed under UV illumination. The sections were subsequently stained immunocyto- chemically with the el-antiserum.

Results

Central nervous system

Pulmonates

Basommatophorans. In B. truncatus, single giant neurons were observed in caudal regions o f bo th the visceral and right parietal ganglia (Fig. 1 A, B). These ganglia also each conta ined 2-5 medium-sized neurons. In addit ion, 3-5 medium sized and a cluster o f small neurons were

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Figs. 5-10. Immunocytochemical staining, with the ~l-antiserum, of neurons and fibers in sections of the central nervous system of two pulmonates (Figs. 5, 6), an opisthobranch (Fig. 7), a prosobranch (Fig. 8), and two bivalves (Figs. 9, 10)

Fig. 5. Eobania vermiculata. A Two intensely stained giant (solid arrows) and a moderately stained small neuron (open arrow) in the subesophageal ganglion, x 125. B Axonal terminals (arrows) on a large neuron in one of the pedal ganglia, x 140. C Dense staining of fibers (solid arrow) in the pedal ganglion and of fibers running in the connective tissue surrounding the ganglia (open arrows). Asterisk Statocyst. x 50

Fig. 6. Helix aspersa. (A, B, E-G) and H. pomatia. (C, D). A Giant neuron (solid arrow) in the subesophageat ganglion and several thick cross-sectioned axons (open arrows) in the intestinal nerve. x 120. B Small neuron in the same ganglion, x 140. C Fluorescent staining of a Br neuron (asterisk) in the subesophageal ganglion. x 75. D Identical section stained with el-antiserum. Note that the same neuron (asterisk) is immunostained, x 75. E Axonal terminals (arrows) in connective tissue surrounding ganglia, x 200. F Axonal terminals in pedal ganglia (arrows). x 35. G Higher magnification showing varicose fibers in neuropil, x 240

Fig. 7A-C. Aplysia t)rasiliana. A Giant neuron (arrow) in the abdominal ganglion, x 50. B Higher magnification showing extensions of the cytoplasm (arrow) 9f the giant abdominal neuron. x 230. C Medium-sized neurons (arrows) in the abdominal ganglion. x 50

Fig. 8A-C. Viviparus viviparus. A, B Neuron (open arrow) in the cerebral ganglion, x 230. C Fibers (open arrows) in the neuropil of the cerebral ganglion, x 230

Fig. 9A-C. Mytilus edulis. A, B Neurons (open arrows) in the abdominal ganglia. A x 230, B x 50. C Fibers in the neuropil of the abdominal ganglion and around an unstained neuron (open arrow), x 230

Fig. 10A-C. Anodonta sp. A, B Neurons (open arrows) in the abdominal ganglia, x 230. C Fibers (open arrows) in the neuropil of the same ganglion, x 230

observed in the cerebral ganglia (Fig. 1 C, D) ; 2 -4 small neurons were observed in the pedal ganglia (Fig. 1 E). Immunopos i t ive fibers were present in the neuropil o f the ganglia and in the connectives, in peripheral nerves, and in the p igmented connective tissue sheath sur rounding the ganglia.

Stylommathophorans. Two to 4 s trongly immunoreac t ive giant neurons were observed in the partially or fully fused visceral and parietal ganglia o f all s ty lommatho- pho ran species examined. The initial axonal segments o f the giant neurons gave rise to numerous secondary branches. Some o f these neurites left the C N S in the subesophageal nerves, whereas others innervated the other ganglia o f the CNS, part icular ly the pedal ganglia. Medium-sized and /o r small neurons, often less intensely stained, were also found in these and in other ganglia. Fur thermore , dense innervat ion was observed in the connective tissue sheath sur rounding the CNS and in the external layers o f b lood vessels, including the anterior aorta.

A. fulica. One giant neuron was observed along the caudal margin o f the right parietal gangl ion near the visceral gangl ion (Fig. 2A). A second giant neuron was located along the caudal margin o f the visceral gangl ion (Fig. 2 B). The axon o f each neuron gave rise to several small axons that b ranched within the neuropil o f these ganglia (Fig. 2C). Several other thick axons projected to other ganglia, or left the CNS via the visceral and parietal nerves. One o f the major visceral nerves (pre- sumably the intestinal nerve) conta ined 5-10 thick axon branches. A total o f 10-15 medium-sized neurons were observed in the visceral and right parietal gangl ion (Fig. 2D). Immunoreac t ive axons appeared to terminate u p o n at least two medium-sized neurons located at the dorsomedia l surface o f the pedal ganglia (Fig. 2 E, F).

Limax maximus. Three giant neurons and 5 8 medium- sized neurons were dispersed within the parietal and visceral ganglia (Fig. 3 A - D ) . Two to four faintly stained medium-sized neurons were present in the pedal ganglia (not shown).

Cepea sp. Two pairs o f adjacent giant neurons were observed. One pair was found in the viscera! gangl ion and the other pair was found in the right parietal ganglion. One member o f each pair stained m u c h more intensely than the other (Fig. 4A, B). Faintly stained small and medium-sized neurons were observed in different ganglia.

Eobania vermiculata. Two giant neurons were observed in the fused visceral/left parietal ganglion. Medium-sized immunoposi t ive neurons were adjacent to these giant neurons (Fig. 5 A). Ano the r giant neuron was observed in the r ight parietal gangl ion (Fig. 5A). N u m e r o u s immunoreac t ive fibers appeared to terminate in the neuropil and a round the cells o f the pedal ganglia (Fig. 5 B, C). Other fibers appeared to terminate in the connective tissue sheath o f the CNS (Fig. 5 C).

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Helix aspersa and H. pomatia. Each of these species pos- sessed two giant immunopositive neurons (Fig. 6A). In H. pomatia, in which LY was injected into Br or Br' before immunostaining, the immunopositivity coloca- lized with the LY fluorescence (Fig. 6 C, D). Some intensely stained small neurons were also observed in these ganglia (Fig. 6B). Numerous immunopositive fibers terminated in the pedal ganglia (Fig. 6F, G) and in the connective tissue sheath surrounding the CNS (Fig. 6 E).

Opisthobranchs

In the abdominal ganglion of A. brasiliana, one strongly stained giant neuron was located along the caudal margin of the right hemiganglion (Fig. 7A). Another 10-15 medium-sized neurons were found in the medial part of the fused ganglion. The cell body of the giant neuron appeared to have numerous small peripheral extensions (Fig. 7 B). Furthermore, intensely stained fibers were observed in the center of the ganglion (Fig. 7C) and in the nerves leaving the ganglion. A. californica showed a similar distribution of less intense immunoreactivity.

Prosobranchs

The cerebral ganglia of V. viviparus contained several small immunopositive neurons and fibers. The neurons (Fig. 8A) were located predominantly near the surface of the ganglia. Immunoreactive fibers were distributed throughout the neuropil of the ganglia (Fig. 8B) and in peripheral nerves. The other ganglia were not studied. Immunoreactivity in the neurons and fibers of N. reticulatus was found to be similar in distribution to that of V. viviparus (not shown).

Figs. 11-17. Immunocytochemical staining, with the el-antiserum, of sections of peripheral organs and tissues of some molluscs. A Auricle; L lumen; P pericardium; Vventricle

Fig. 11. Bulinus truncatus. Nerve fibers (open arrows) innervating muscle fibers in the auricle of the heart, x 350

Fig. 12. Achatina fulica. Nerve fibers (solid arrow) terminating among muscle fibers in the auricle of the heart. Note the bundle of fibers (open arrow) running in the pericardium, x 25

Fig. 13. Cepea sp. Nerve fibers (arrow) terminating among muscle fibers in the auricle of the heart, x 35

Fig. 14A-C. Eobania vermiculata. A Nerve fibers (solid arrow) terminating among muscle fibers in the auricle of the heart (open arrow). Nerve fibers running in the pericardium, x 55. B Higher magnification of terminals among auricular muscles, x 200. C Ax- onal terminals (open arrows) within peripheral layers of the anterior aorta, x 55

Fig. 15A, B. Helix aspersa. A Nerve fibers (arrow) terminating among muscle fibers in the auricle of the heart, x 70. B Bundle of fibers in the pericardium, x 166

Fig. 16A, B. Aplysia californica. Axonal terminals (arrows') among auricular muscle fibers. A x 50, B x 125

Fig. 17A, B. Mytilus edulbx. Gill with gill-plates (solid arrows). Note that muscle that are located next to the gill-plates contain immunoreactive fibers (open arrows), x 250

Bivalves

The CNS of M. edulis contained 40-50 small immunopositive neurons, located peripherally in the abdominal ganglia (Fig. 9 A, B). Many immunopositive fibers were observed in the neuropil of these ganglia; some of the fibers appeared to terminate upon larger neurons (Fig. 9 C). Fibers were also observed to leave the ganglia via peripheral nerves. Several positive neurons were also observed in the cerebral ganglia. Immunoreactivity in the neurons and fibers of Anodonta sp. was similar to that observed in M. edulis (Fig. 10 A -C).

Peripheral organs (heart, gill)

Numerous axonal terminals were observed in the auricles of the hearts of all pulmonates and opisthobranchs. Fur- thermore, the pattern of heart innervation was identical in all pulmonate species (Figs. 11-15). A bundle of immunopositive fibers in the pericardial branch of the intestinal nerve traversed the pericardium (cf. Figs. 12, 14A, 15 B) to the venous side of the auricle of the heart. At this position, immunopositive fibers entered the pericardium and terminated among the auricular muscle fibers (Figs. 11-15). In most species, (e.g., in E. vermieu- lata, Fig. 14A, and in H. aspersa, Fig. 15A), some nerve fibers were also observed in the ventricular valves, but no immunopositive fibers were ever found to enter the ventricle proper. Immunoreactive fibers were generally observed in the peripheral layers of the aorta (Fig. 14C). In the opisthobranch heart, immunopositive nerve fibers were also observed along among the auricular muscle fibers (Fig. 16 A, B), and not among the ventricular muscles.

In prosobranchs and bivalves, no immunostaining was observed in the heart. However, in the bivalve M. edulis, varicose immunopositive fibers were present in muscle fibers adjacent to the gill plates (Fig. 17A, B). The gills of the other animals were not studied.

Discussion

The results demonstrate that the el-antiserum, raised against one of the neuropeptides of the VD1/RPD2 system of L. stagnalis, recognizes neurons not only in a variety of pulmonates, but also in opisthobranchs, prosobranchs and bivalves. This finding suggests the evolutionary conservation of neurons that may contain structurally related neuropeptides. Although cross-reactions with unrelated molecules may have contributed to our results, several additional observations support hypotheses of homologous cells and peptides. First, a single giant neuron and several smaller neurons were stained in the abdominal ganglion of both Aplysia species studied. Based on its size and location, the giant neuron is most probably RI5. R15 and several smaller neurons in the abdominal ganglion have previously been shown to express a gene that is homologous to the VD1/RPD2 gene of L. stagnalis (Buck et al. 1987; Bogerd et al. 1993).

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Furthermore, immunoreact ive giant neurons in the other pulmonates have soma, locations and axonal branching patterns similar to those of VD1 and RPD2. In the clear- est example, LY injections demonstrate that the Br and Br' neurons of H. pomatia react with the cO-antiserum. In addition to their similar morphology and biochemis- try, these cells also share several physiological features with VD1 and RPD2 (Pin and Gola 1983), and with R15 (e.g., Alevizos et al. 1991). Based upon somatic position and neuritic branching patterns, the giant neurons found in A. fulica most probably are V I N and PON (Furakawa and Kobayashi 1987). Chase and G o o d m a n (1977) have previously argued that P O N (termed RPr l 1) is probably homologous to R15, based upon similarities in location and electrophysiology. Thus, our work adds molecular evidence to previous suggestions that certain parietal and visceral cells in pulmonates are homologous to R15 in Aplysia (see also Gainer 1972; Sakharov 1976). It further suggests possible evolutionary conservation of several smaller cells that express peptides reactive to the e l -ant iserum. In the basommatophoran , B. truncatus, the distribution of these neurons is similar to that de- scribed for L. stagnalis (Kerkhoven et al. 1992). In sty- l ommatophoran pulmonates and in opisthobranchs, however, more evidence is needed to establish homologies of the unidentified smaller neurons between species. Furthermore, prosobranchs and bivalves do not possess giant (polyploid) neurons and therefore an extention of homologies within these animals also awaits further research.

The function of the immunoreact ive peptides is un- clear, but certain clues emerge f rom comparisons of peripheral projections of immunoreact ive fibers. In all pulmona te and opis thobranch species studied, the auricle (but not the ventricle) appears to be innervated by immunoreact ive fibers. In Lymnaea, V D , / R P D 2 project to the heart (Kerkhoven et al. 1991). In Aplysia, the R15

peptide has been detected in the auricle and ventricular valve, but not the ventricle itself (Skelton and Koester 1991). These findings are therefore consistent with both a cardiovascular and a renal function for the peptides, since a hemolymph u!trafiltrate drains f rom the auricle to the kidney (Andrews and Little 1972; Boer et al. 1973; De With 1980). Certain ~ neuropeptides, indeed, seem capable of modulat ing heartbeat in Lymnaea (Bogerd 1992), whereas crude extracts of R15 seem to modulate osmoregulat ion in Aply~ia (Kupfermann and Weiss, 1976). However, innervation of the heart has not been found in prosobranchs and in bivalves. On the other hand, the observation that the gill receives innervation in M. edulis indicates similarity in a second possible function for the VD1/RPDz-sys tem: the regulation of respiration. It has been shown that VD, and RPD2 are involved in the regulation of respiration (Van der Wilt et al. 1987). In Aplysia, R15 has also been shown to influence respiratory functions (Alevizos et al. 1991). Fu- ture work must resolve if the various e-peptides that have been identified in Lymnaea and Aplysia have unrelated functions, or whether the different peptides all play an integrated role in the same physiological phenome- non. Future work must also determine the extent to

which each function is conserved across molluscan evo- lution. Such research will be guided by the identification of seemingly homologous cells and their targets as pre- sented in this study.

Acknowledgement. We thank an anonymous reviewer of an earlier version of this article for extensive corrections and suggestions.

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