Comparative Anatomy ofPACAP-Immunoreactive Structures inthe Ventral Nerve Cord Ganglia ofLumbricid Oligochaetes

LASZLO MOLNAR,a EDIT POLLAK,a AKOS BOROS,a DORA REGLODI,b

ANDREA TAMAS,b ISRVAN LENGVARI,b AKIRA ARIMURA,c

AND ANDREA LUBICSb

aDepartment of General Zoology, University of Pecs, 7624 Pecs, HungarybDepartment of Anatomy, Neurohumoral Regulations Research Group of theHungarian Academy of Sciences, University of Pecs, 7624 Pecs, HungarycUS-Japan Biomedical Research Laboratories, Tulane University, New Orleans,Louisiana 70037, USA

ABSTRACT: By means of a whole mount immunocytochemical ap-proach, the distribution patterns of pituitary adenylate cyclase-activatingpolypeptide (PACAP)-27 and PACAP-38 were identified in the ventralnerve cord (VNC) ganglia of the earthworms Eisenia fetida and Lumbri-cus terrestris. Each PACAP form appears to occur in a distinct neuronpopulation. Positions of these populations, as well as numbers and sizesof the constituting neurons do not essentially differ between the twospecies. The data suggest that in Lumbricid Oligochaetes, PACAP-27and PACAP-38 neuron populations may mediate distinct physiologicalprocesses.

KEYWORDS: earthworm; Eisenia fetida; Lumbricus terrestris

INTRODUCTION

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly con-served neuropeptide, with only one to four amino acid differences betweendifferent vertebrate species.1 Its presence has also been shown in invertebrates,from protozoa2 to protochordata.3 In the latter group PACAP-27 shows 90%similarity to human PACAP.3 Except for Drosophila, in which PACAP maybe involved in synaptic activity modulation4,5 and memory processes,6,7 there

Address for correspondence: Dora Reglodi, M.D., Ph.D., Department of Anatomy, University ofPecs, 7624 Pecs, Szigeti u 12. Hungary. Voice: +36-72-536001; ext.: 5398; fax: +36-72-536-393.

e-mail: dora.reglodi@aok.pte.hu

Ann. N.Y. Acad. Sci. 1070: 427–430 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1317.056

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FIGURE 1. Whole mount immunocytochemistry of distribution pattern of PACAP-38in VNC ganglia of Eisenia fetida (A) and Lumbricus terrestris (B). Anterior on the top.Scale bar 100 �m (A) and 250 �m (B). Note small numbers of neurons located at the levelof the first, second, and third segmental nerves.

are relatively few data about PACAP functioning in invertebrates. PACAP-likeimmunoreactivity is present in annelids, and by means of radioimmunoassay,PACAP-27 has been proposed to represent the major form of the peptide in theoligochaete earthworm, Lumbricus polyphemus.8,9 However, the exact anatom-ical positions of neurons expressing PACAP-38 or PACAP-27 have not beenidentified. The aim of the present article was to establish the three-dimensionaldistribution of PACAP forms in the ventral nerve cord (VNC) of oligochaeteworms Eisenia fetida and Lumbricus terrestris by means of a whole mountimmunocytochemical approach.

MATERIALS AND METHODS

All experiments were carried out on 5–5 sexually mature specimens of Eise-nia fetida and Lumbricus terrestris (Annelida, Oligochaeta) kept in a standardbreeding stock at our department. After anesthesia with chilling and carbondioxide,10 the VNC was dissected from the anterior 15–20 body segments andfixed in freshly prepared 4% ice-cold phosphate-buffered paraformaldehyde(pH 7.2) for 4 h. For whole mount immunocytochemistry, VNC ganglia weretreated by immersion in 0.5% Triton-X 100 in 0.1 M phosphate-buffered saline(PBS), and then incubated with either PACAP-27 (No. 88,121–5)11 or PACAP-38 (No. 92,112–6)12 (1:500–1:1000) antisera, immunostained by avidin-biotin

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FIGURE 2. Whole mount immunocytochemistry of distribution pattern of PACAP27in VNC ganglia of Eisenia fetida (A) and Lumbricus terrestris (B). Anterior on the top.Scale bar 100 �m (A) and 250 �m (B). Note high numbers of neurons at the ventrolateraland lateral parts of the ganglion and a pair of large neurons behind the first segmentalnerves.

horse radish peroxidase staining kit (Sigma, Budapest, Hungary) and devel-oped with 0.03% 3,3’-diaminobenzidine. After immunostaining and thoroughwashing in PBS, VNC ganglia were cleared in glycerol and observed with aNikon Eclipse 80 microscope applying brightfield and/or Nomarski illumina-tion.

RESULTS AND DISCUSSION

Results were essentially the same for each of the two earthworm species.Both PACAP-27 and PACAP-38 immunoreactivities were identified in theVNC, in fully separate neuron populations. PACAP-38-containing neuronswere mainly located ventromedially and ventrolaterally at the level of seg-mental nerves. Neither somata of dorsal giant axons and giant motoneurons,nor neurosecretory cells13 were found among stained cells (FIG.1). In contrast,PACAP-27-labeled neurons mainly occurred in the ventrolateral and lateralparts of ganglia, except for a pair of large somata behind the first segmentalnerves and a few small neurons situated ventromedially (FIG. 2). The number ofPACAP-27-labeled neurons was much higher (100–108 neuron per ganglion)than that of PACAP-38-stained ones (26–30 neuron per ganglion), which isconsistent with previous data indicating that PACAP-27 is the predominant

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PACAP form in the earthworm nervous system.9 The size and the anatomicalposition of small-labeled neurons strongly suggest that they are interneuronsforming fine fibers in polysegmental interneuronal tracts.13

The anatomical positions of PACAP-labeled somata do not resemble the pat-tern of any other neurons identified in Eisenia fetida and Lumbricus terrestris,suggesting that PACAP27 and PACAP38 are expressed in distinct neuron pop-ulations the members of which mediate specific physiological processes.

ACKNOWLEDGMENTS

This work was supported by the National Science Research Fund (OTKAT026652, T046589, F048908) and the Hungarian Academy of Sciences.

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