Nematode neuropeptides: Localization, isolation and functions

R~iews 37 Lewis, F.A. et al. (1987) lntraspecifie cross-protection in mice immunised with irradiated Schistosoma mansoni cercariae. I. Parasitol.73, 787-791 38 Bickle, Q.D. and Doenhoff,M.J. (1987) Comparisonof the live vaccine potential of different geographic isolates of Schistosoma mansoni. ]. Hehniathol. 61,191-195 39 Lewis, F.A., Hieny, S. and Sher, A. (1985) Evidence against the existence of specific Schistosmna mansoni subpopulations which are resistant to irradiated vaccine-induce immunity. Am. ]. Trop.Med. Hyg. 34, 86-91 40 Simpson,A.J.G.et al. (1985)Antibodyresponseagainst schistosomulumsurfaceantigens and protective immunity following immunizations with highly irradiated cercariae of Schistosoma mansoni. ParasiteImmonol. 7,133-152 41 LoVerde,P.T. et al. (1985) Evidence for host-induced selection in Schistosoma mansonL I. Parasitol.71,297-301 42 Br6mond, P. et aL (1993) Experimental host-induced selection in Schistosmna mansoni strains from Guadeloupe and eom-

parison with naturalobservations.Heredity 70, 33-37 43 Simpson,A.J.G.(1987)'11~influ~¢e of molemlar~ i t y in helminth identifw.ation,protective immunity and immunodiagnosis, int. I. Parasitol.17, 69-77 44 Richter, D., Ham, D.A. and Matuschka, F-R. (1995)The irradiated cercariae vaccine modeh looking on the bright side of radiation. Parasitol.Today 11, 288-293 45 Higgins-Opitz, S.B. and Dettman, C.D. (199l) "[he infection characteristics of a South African isolate of Schlstosoma mansoni: a comparison with a Puerto Rican isolate in BALB/e mice and Mastomys coucha. ParasitoLRes.77,142-151 46 Quinnell,R.J.,Behnke,J.M. and Keymer,A.E.(1991)Host specificity of and cross-immunitybetween two strains of Heligmosomoides polygyrus. Parasitology102,419--427 47 Chapman, C.B., Rajasekariah, G.R. and Mitchell, G.F. (1981) Clonal parasiles in the analysis of resistance to reinfection with Fasciola hepatica. Am t- Trop. Med. Hyg. 30, 1039-1042

A British Society for Parasitology Symposium entitled Molecular Biochemistry and Physiology of Helminth Neuromuscular S y s t e m s organized by D.W. Halton and R.J. Martin, will be held 18-19 September 1996 at the City University, London, UK. The two articles that follow will whet the appetite of those interested in this topic.

N e m a t o d e Neuropeptides: Localization, Isolation and Functions D.J.A. Brownlee, I. Fairweather, L. Holden-Dye and R.J.Walker Historically, peptidergic substances (in the fonn of neurosecretions) were Ihzked to moulting in nematodes. More recently, there has been a renewal of interest in nematode neurobiology, initially triggered by studies demonstrating the localization of peptide immunoreactiviti~ to the newous system. Here, David Brownlee, lan Fairweather, Lindy l-~ldeaDye and Robert Walker will review progress on the isol~#ion of nematode neuropeptides and efforts to unravel their physiological actions and inactivation mechanisms. Future avenues for research are suggested and the potential exploitation of peptidergic pathways hi filture therapeutic strateg&~ highlighted. The nervous system of parasitic nematodes is e::ceptionally well-defined in terms of the number, location and projections of the small number of neurones involved in regulatory behaviours vital to their survivaP -3. This is largely because of the vast amount of information available on the free-living nematode Caenorhabditis elegans 4, and the neuroanatomical similarity between this species arid parasitic species such as Ascaris suum. However, functional information has not paralleled these advances, and it is only recently that the chemical complexity of the nematode nervous system has become apparent s. This fresh insight h~.s resulted from s~c,dies on the peptidergic component of the nervous system and the isolation of an increasing number of peptides from nematodes. This review David Brownlee and lan Fairweather are atthe School of Biology and Biochemistry,The Queen's University of Belfast, Belfast, UK BT7 INN. D.J.A. Brownlee is also, with Lindy Holden-Dye and Robert Walker, at the Department of Physiologyand Pharmacology, School of Biological Sciences, University of Southampton, Southampton, UK SO 16 7PX. Tel: +44 1232 2451:3:3 x 2298, Fax: +44 1232 236505, e-mail: i.fairweather~qub.ar.uk

Parasitology Today, vol. 12, no. 9, 1996

will summarize data on the distribution of n e u r o p e p tides in the nematode nervous system, survey p r o gress in the isolation of peptides 'native' or e n d o g e n ous to nematodes and examine evidence supporting physiological roles for peptides in these organisms. Although immunoreactivities to a variety of peptides have been localized in the nervous system, all the endogenous peptides isolated to date are FMRFamiderelated peptides (FaRPs). Consequently, the main focus of the review will be on this family of peptides. The first indication that nematodes possess p e p tidergic nerve cells came in 1958 with the ascertainment of paraldehyde fuchsin-posifive neurosecretory cells in Ascaris 6. Neurosecretory cells have been identified in a number of trematodes and the results supported by ultrastructural evidence of typical densecored neurosecretory vesicles7. The main function attributed to the neurosecretions was in the ecdysis phase of moulting. Work on the cod worm, Phocanenta decipiens, by Davey and colleagues (reviewed in Ref. 7) showed that a cycle of secretory activity in nerve cells belonging to ganglia associated with the anterior nerve ring was linked to the moulting of the third stage larva. The cells were envisaged to produce an "ecdysial" hormone which acts on the excretory cell to activate and bring about the release of enzymes comprising the moulting or exsheathing fluid. The fluid is secreted via the excretory duct into the space b e t w e e n the old and new cuticles and serves to digest the o l d cuticle 7. The identity of the presumptive "ecdysial~ hormone or, indeed, other neurosecretions is u n k n o w n L¢ be

Copyright© 1996,Elsc~erScienceL~dAllnghtsi

iReviews

rig; I. ~.ompos~te contocal scanning laser m|crograpn ~.~Lrl) Ot me anterior ,!erve ring (anr), showing SALHFamide immunoreactivity (SI-IR) in nerve cell bodies belonging to the ventral ganglion (vg) and anterior (alg) and posterior (pig) lateral ganglia. SI-IR is also evident in nerve fibres constituting the amphidial commissure (ac); pn, papillary nerve fibres. This is a composite CSLI image of 21 optical sections taken at 2 Fm interva s through the specimen. Sea e bar = 50 p.m.

of the neurosecretory cell has been superseded by the term 'peptidergic neuron'. Immunocytochemical studies since the late 1980s, involving the application of antisera raised to a wide range of vertebrate and invertebrate peptides, have led to a resurgence of interest in the nematode nervous system. Moreover, it has provided some clues as to the nature of the peptidergic molecules present in nematodes. The studies have established that nematodes possess an extensive and varied peptidergic component of their nervous system: a component which, in ~em~s of putative transmitter molecules, predominates over 'classical' neurotransmitters, such as acetylcholine. The patterns of immunostaining within the central nervous system (CNS) vary from peptide to peptide, and among the main immunoreactivities demonstrated are molecules resembling vertebrate peptides belonging to the neuropeptide Y superfamily and the gastrin/cholecystokinin family, and the invertebrate peptide, FMRFamide 5.s.9 (Fig. 1). It has been estimated that up to 75% of nerve cells in Ascaris are FMRFamide-

Table I. Primary structure, sequence comparison and actio;, of FMRFamide-related peptides (FaRPs)from the nematodes Ascaris suum, Haemonchus contortus, Caenorhebditis elegans and PanagreUus ~edivivus Species Parasitic A. . . . .

H. contortus Free living

Celegans

P. redivivus.

Peptide

Sequence

AFI AF2 AF3 AF4 G AF5 S AF6 AF7 AF8 c AF9 G AFI0 G F G D E M AFII S D I G I AFI2 F G D E M AFI3 d S AFI4 d AF20 d AF2 CFI CF2 CF3 CF4 CF5 CF6 CF7 AF2 PFI PF2 PF3 PF4 AF2

Ref. Action on Ascaris (sequence) body wail muscle

R,f, (a,ction) 18,31 19, 33 b 20 b b

35 36

K N H E F

IL

R E ;

A V P G V D V P G V G K P T F F A G P R F K S A Y L G P R P S M P G V S E P N F S M P G V D M F G V S M P G V G M P G V K H E Y

L L I I I M L L L L L L L L

R R R R R R R R R R R R R R

F F F F F F F F F F F F F F

NH z NH z NH z NH z NH z NH z NH= NH z NH z NH2 NH 2 NH z NH 2 NH z NH z NH 2

18 19 20 20 20 20 20 20 20 20 20 20

28

Excitatory Biphasic Excitatory Excitatory W e a k inhibition No effect N.D. Excitatory N.D. N.D. N.D. N.D. N.D, N,D, N.D. Biphasic

L L L L L L L L L L M I L

R R R R R R R R R R R R R

F F F F F F F F F F F F F

NH 2 NH z NH 2 NH 2 NH z NH 2 NH 2 NH z NH 2 NH 2 NH 2 NH z NH 2

21,22 21,22 21,22 21,22 21,22 21,22 21 23 24 24 26 27 25

Inhibitory inhibitory N.D. N.D. N.D. Noeffect N.D. Biphasic Inhibitory Inhibitory Excitatory Inhibitory Biphasic

S S A S A S G A A A A G S

D D Q D D

D K S D S A D K K K

P P P P P P P H P P S P H

N N N N N N N E N N A N E

F F F F F F F Y F F Y F Y

26, u

19,33

b 19,33 35 36 26, b 27, 32 19,33

~Key: black (N.D. not determined); red, excitatory; blue, inhibitory; green, no effect, bL. Holden-Dye, unpublished. dA S Ed son, unpub shed [GenBank (1994), accession number-U15279]. ~Causespotent inhibition of Ascaris pharynx. 344

Parasitology Toclay, vol. 12, no. 9, 1996

immunoreactive~,9. The predominance of FMRFamide immunoreactivity is not surprising, given that the endogenous peptides isolated to date are FaRPs (Table 1). The overlap of staining and consequent potential co-localization of peptide immunoreactivities observed at the optical level8,9 has been confirmed by immunogold labelling at the subcellular levello.H. The neuronal location of peptides within the CNS argues for a neurotransmitter and/or neurom~dulator role for the peptides. Supporting physiological data will be discussed below. In the peripheral nervous system (PNS), innervation of sense organs (papillae, amphids, phasmids, deirids) by peptide-immunoreactive nerve fibres is a feature of Ascaris and other nematodes9,12,13 (Fig. 2a). It confirms previous neurosecretory studies which indicated that the majority of anterior sensory cells in Ascaris are fuchsinophilic14. The results suggest that peptides are involved in sensory perception and transduction, especially mechano- and chemoreception 12. Innervation of various reproductive s0.1actures (eg. vulva, vagina, copulatory spicules) by peptidergic nerve elements has led to the suggestion that peptides may be involved in copulatory behaviour, egg laying and other aspects of reproductive biology9,~2,~5,~6(Fig. 2b). There is a significant paptidergic component in the enteric nervous system (ENS) 17, indicating a possible role in feeding activity; this area will be discussed separately below.

Isolation of endogenous peptides and cloning of peptide genes Work on nematode peptides has now progressed beyond the localization of peptide immunoreactivities to the isolation and sequencing of peptides endogenous to nematodes themselves The first endogenous peptide, a FMRFamide-related peptide from Ascaris (designated AF1 from Ascaris FMRFamide-like peptide 1) was isolated in 1989 (Ref. 18). This was followed, in 1990, by a second peptide, AF2, from Ascaris ;9. This style of annotation has been followed for other peptides isolated from Ascaris and for peptides isolated from C. elegans (CF1-7) and Panagrellus redivivus (PF1--4). To date, 22 confirmed peptide sequences have been published: ten from Ascaris ls-2°, seven from C. elegans21-23, five from P. rediviw~s24-27 and one from Haemonchus contortus 2s (Table 1). A number of putative (unconfirmed) peptide sequences have been reported; they are also listed in Table 1. As is evident from the Table, a number of peptides are common to more than one species of nematode. For example, C. elegans and P. rediviwts possess the peptides SDPNFLRFamide (PF1, CF1) and SADPNFLRFamide (PF2, CF2) 2 ~ 4 . The peptide originally isolated from Ascaris as AF2 (KHEYLRFamide) 19 is also present in C. elegans 23, P. redivivus 25and H. contortus 28. Finally, KSAYMRFamide is common to P. redivivus (as PF3)26 and Ascaris (as AF8) 2°. The great majority of the peptides were Parasitology ~bday, voL 12, no. 9, 1996

isolated by conventional chromatographic techniques. An alternative, molecular approach was adopted for C. elegans, resulting in the isolation of a gene, tip-l, that encodes seven FMRFamide-like pepti~.ies2L FIp-1 produces two transcripts, A and B, with transcript B encoding a peptide not found in transcript A. The transcripts are produced by alternative splicing of the tip-1 gene and this, together with the encoding of multiple peptides by the gene, generates neuropeptide diversity. The organization of the gene is similar to that of genes encoding FMRFamide-like peptides in other organisms29, but is unusual in that only FLRFamide-related, not FMRFamide-related, peptides are encoded 21. A subsequent chromatographic study was carried out to confirm whether the putative peptides encoded by tip-1 are produced in vivo: six of the seven predicted peptides were isolated22. Expression studies in worms carrying lacZ reporter constructs have shown that tip-1 is expressed in RFamide-immunoreactive interneurons in the head region. To charac' terize the function of tip-l, a tip-1 deletion mutant, or 'knockout', was isolated. A number of phenotypes have been correlated with the tip-1 deletion: they include hyperactivity, nose-touch insensitivity, defects in osmolarity detection and resistance to serotonininduced egg-laying (C. Li, pers. commun.). A gene, Cv-flp-1, sharing strong sequence homology with the tip-1 gene in C. elegans has been identified in the related species, C. wdgaris 3°. The gene also produces two transcripts through alternative splicing, and encodes a set of peptides identical to that in the flp-~ gene3°. More recently, other genes encoding FaRPs have been identified in C. elegans: they areflp-2 (PIRFamide-containing peptides), tip-3 (FIRFamide-containing peptides) and tip-4 (TMRFamide-containing peptides) (C. Li, pers. commun.). A partial cDNA sequence encoding FMRFamide-like p e p t i d ~ inAscaris has been isolated: it contains novel ~ b h C ~ fo~ AF13, AF14 and AF20, in additi0n t6 ~ d ~ AF3, AF4 and AF10 previously d ~ r i ~ (A!S Edi~n~ unpublished: NCBI GenBank D a t a b a ~ a c c ~ i 6 h no!

3,IS

Reviews Motoneuronee

Somatic mascle

Fig. 3. A stylized diagram of the ne,romuscular junction in the Ascaris motor nervous system depicting the excitatory motor neurones (red) releasing acetylcholine (ACh) and the inhibitory motor neurones (blue) releasing GABA on to the somatic musculature to elicit contraction and relaxation, respectively. The FaRPsfor which the site of action has been established (as either presynaptic or postsynaptic) are shown in the small boxes. It is likely that this will become increasingly complex in the near future. For example, AF3 and AF4 cause muscle contraction, and AF2 can cause muscle relaxa'.ion (in addition to contraction), but the mechanisms of action have not been unequivocally established. Key: a postsynaptic effect on the muscle is indicated with a black arrow, a presynaptic action on the motor nervous system is indicated by either a red arrow for an excitatory action (eg. AF2 stimulates ACh release by a presynaptic action) or a blue arrow for an inhibitory action (eg. PFI inhibits ACh release by a presynaptic action).

U15279). It is the roles of some of these endogenous peptides in the neurophysiology and pharyngeal pumping behaviour of nematodes that will be discussed below. The specific location of individual peptides and their receptors within the nervous system remains one of the most important unanswered questions in nematode neurobiology. Physiological roles for FaRPs in the n e m a t o d e nervous system The biological activity of the FaRPs has been assessed in Ascaris as it is the nematode that is most amenable to electrophysiological techrJques. The studies are far from complete, but already some interesting principles are emerging. First, many of the peptides are very potent at eliciting their actions, at least two orders of magnitude more potent than the 'classical' transmitters acetylcholine (ACh) and ~/-aminobutyric acid (GABA). (Acetylcholine is the excitatory transmitter at the Ascaris neuromuscular junction and causes muscle centraction, whereas GABA is the inhibitory transmitter and causes muscle relaxation.) Second, it is evident that there is more than one excitatory, and more than one inhibitory, peptidergic mechanism present. Third, the peptides studied so far interact both presynaptically and postsynaptically at t h e neuromuscular junction. Fourth, some of the FaRPs ~licit fast responses within seconds that reverse

rapidly o1~ washing, whereas others have very longlasting actions of more than an hour's duration, even after just a single brief exposure of the tissue to the peptide. Lastly, the nematode FaRPs are very closely structurally related and small changes in peptide sequence can have a profound effect on biological activity [eg. KNEFIRFamide (AF1) is predominantly excitatory31 on Ascaris body wall muscle whereas KPNFIRFamide (PF4) is inhibitory27.32]. The motor nervous system. Neuroactive peptides can act to modify nematode physiological processes either within the central ganglia, on the nerve terminals of motor or sensory neurones, or directly on effector organs, such as pharyngeal muscle or ~owatic body wall muscle. The effect of the peptides on the latter tissue has been the most comprehensively studied and the data are summarized in Table 1 and Figs 3, 4. The FaRPs which have an excitatory action on Ascaris body wall muscle are AF1 (Ref. 31), AF2 (Ref. 33), AF3 (L. Holden-Dye, unpublished), AF4 (Ref. 20) and AF8 (also known as PF3) (Ref. 26). They all elicit contraction of Ascaris vr~uscle, though the responses are qualitatively different (Fig. 4, a-d). The responses can be broadly classified as either strong tonic contractions or an increase in the frequency and amplitude of phasic muscle contractions and relaxations. The peptides themselves may also be grouped into two classes according to their sequence similarity. Thus, AF1, AF2 and AF8 (Group A) are all heptapeptides with an N-terminal Lys, whereas AF3 and AF4 (Group B) are eight and nine residues long and share an identical C-terminal of -PGVLRFamide. The Group B peptides and AF8 cause a long tonic contraction of Ascaris muscle. In contrast, the Group A peptides, AF1 and AF2, elicit both tonic and phasic responses. The action of AF2 is particularly remarkable as the predominant action is an increase in muscle activity which is evoked by low (nanomolar) concentrations of the peptide and may last for up to two hours 19,33.At the cellular level, AF2 has a number of different effects, but the action that most closely parallels the time-course of the increase in phasic activity is the ability of the peptide to act postsynaptically to the neuromuscular junction to increase action potential generation in the muscle syncytium 33. Several lines of evidence suggest that AF2 may be of fundamental importance to nematodes. For example, a monoclonal antibody that distinguishes between AF1 and AF2 has been used to show that AF2 is present in a much larger population of neurones than is AF1 (Ref. 34); immunostaining in H. contortus using a FaRP antibody is preferentially blocked by AF2 (Ref. 28); biochemical isolation indicates that it is a relatively abundant peptide and AF2 has been identified in four species of nematode 19,23,25,28.AF2 also increases phasic activity in isolated muscle strips from the parasitic nematode, Ascaridia galli (L. Holden-Dye, unpublished) which suggests that AF2 may subserve the same physiological role in different species of nematode. The only e~ldogenou~ Ascari~ peptide so far reported to cause Ascaris muscle rel~×ation is AF2 (Ref. 19). The relaxation is brief, precedes the excitatory effect of the peptide ~ d is not observed consistently33. The other inhibitory peptides, PF1, PF2 (Ref. 24) and PF4 (Ref. 27), have not yet been identified in Ascaris. However, because all these peptides have a potent action on Ascaris . . . . Parasitology Today, vol. 12, no. 9, 1996

Reviews ............. muscle, it is most likely that the Ascaris motor nervous system possesses peptides with similar or identical sequences. In this respect, it is interesting to note the structural similarity between AF5 and PF4; also, AF11 and PF1 share a c o m m o n ~dF1 C-terminal sequence, PNFLRFamide, and m a y be species homologues 20. The mechanisms of action of PF1, PF2 and PF4 have been investigated in some detail, with the result that it is quite clear that there are at least two distinct inhibitory peptidergic mechanisms in the Ascaris motor nervous system. The first of these is e f activated by either PF1 or PF2 and elicits a long-lasting flaccid paralysis of the muscle (Fig. 4e). This effect is quite unlike the fast, chloride-mediated inhibition caused by GABA 35. PFI At a cellular level, these peptides cause a muscle cell hyperpolarization and have also been s h o w n to have both presynaptic and postsynaptic actions36. Thus, PF1 acts both Fig. 4. Effects of neuropeptides on the tension of Ascaris somatic muscle. Strips o, to inhibit the release of the excitaAscar/s dorsal muscle (I cm in length) were suspended in an organ bath and connected ~:ory neurotransmitter acetylcholine to an isometric transducer. Acerylcholine or peptides were added to the bath and the effect on muscle tension recorded: 30 s application of 10 p.H acetylchoEne (open a n d to inhibit the response of the triangle). Application (for 5 rain) of I p.M of each of the p e p t ~ (closed triangles), a s muscle to acetylcholine. To what indicated (with the exception of PF4 which was only applied for 2 rain). Vertical scale extent each of these events is involved bars = I ~, horizontal scale bars = 5 rain. in irtediating the muscle paralysis is unclear. However, nanomolar concentrations of PF1 inhibit the muscle contraction caused by acetylcholine and the available evidence suggests that an important site of action of PF1 is on the muscle to inhibit excitation-contraction coupling ~. PF4 h a s a strikingly different m o d e of inhibitory action to PF1 (Ref. 27) (Fig. 4f). The time-course of the muscle relaxation caused by PF4 is m u c h shorter, minutes rather than hours, and its action more closely resembles that of GABA. However, although (like GABA) the response is dependent on chloride ions, it does not involve the release of GABA 32. Rather, PF4 acts postsynaptically to open a GABA-independent chloride channel a n d causes a potent and marked inhibition of muscle contraction. Thus, at the Ascaris neuromuscular junction there is functional evidence for at least four peptidergic modulatory effects, two excitatory and bvo inhibitory. One obvious question that arises is ' w h y does such a simple organism possess such a complex array of cell signalling molecules?' By analogy with other invertebrate systems, neuropeptides m a y fulfil a neurohormonal role within neuronal networks to modulate or generate rhythmic activity especially important for stereotyped behaviours such as locomo,ion or feeding. The action of AF2 on Ascaris muscle is consistent with such a role for this peptide within the motor nervous system. However, this does not rule out the possibility that peptides m a y also act as co-transmitters. Indeed, the fast inhibitory action of PF4 on Ascaris somatic muscle is more akin to a role as co-neurotransmitter than as a hormone. The enteric nervous system: pharyngeal pumping behaviour. The ENS controls the vital function of pharynParasitology Today, vol. 12, no. 9, 1996

Reviews iil ........

Anterior

p

Ipn

,,

n3~

\ P-I

pharynx (P) just distal to the anterior nerve ring. A dense plexus arrows) shows immunoreactivity to SALHFamide (SI) (a). The ~rve processes emanating from the lateral longitudinal pharyngeal ior region of pharynx (13) (b). SALMFamide-immunoreactivity is "nissure (mc) and the dorsal pharyngeal nerve (dpn) tract which res. Exten=ive innervation of the pharyngeal surface in the region hal interface (P-I) consists of fine plexiform nerve plexuses (np). trve cell body (ncb) situated at the posterior region of the dorsal ~ct. Diagrammatic representation (not to scale) showing the dis• pharyngeal enteric nervous system (ENS) of Ascaris (c): dpn, tract; Ipn, lateral pharyngeal nerve tract; mc, main commissure; forked nerve processes; n2 and n3, bushy nerve processes; np, ; I, intestine; P-I pharyngeal intestinal valve (~osition of tube for .=alpressure changes). Inset to (c) shows a magnification of a nerve cell body in the posterior region of the pharynx, Note the extensive neuronal branching in this region. Scale bars = 100/~m (e), 50 v.m (b).

radial muscle which contracts to allow the lumen to fill with liquid and suspended food particles. Rapid relaxation of the pharyngeal muscle causes an increase in pressure, which is sufficient to overcome the high 'internal hydrostatic pressure within the worm, and forces ingested food into the intestine via the pharyngeal intestinal valve. Although there appears to b~' a certain degree of autonomy in the activity of the pharynx, the ENS is linked to the ventral portion of the CNS via two interneurones; they are known as the subventrai nerves17Z7,38. The EN.q is complex, involving cholinergic, serotoninergic and peptidergic nerve elements17. In addition, there is evidence for glutamatergic and GABAergic involvement in pharyngeal functioning (D.J.A. Brownlee, unpublished). In Ascaris, the ENS consists of three main longitudinal nerve tracts (partially embedded within the pharyngeal syncytium) (Fig. 5), cross-connecting commissures and associated nerve cell

bodies and fibres~7(Fig. 6). Along the pharynx at least three types of branched nerve processes are evident (Fig. 7). The processes arise from the lateral longitudinal pharyngeal nerves and ramify across the pharyngeal muscle. Such a widespread innervation may be to attain a more complete innervation of the syncytial muscle and this is important in co-ordinating the muscular and glandular activity of the pharynx during feeding. Immunoreactivifies to a number of peptide-like molecules have been !,ocalized within the ENS: they include pancreatic polypeptide (PP), peptide YY (PYY), gastrin and the invertebrate peptides FMRFamide and SALMFamide ($1)17,39. The ENS of Ascaris is the only component of the nervous system that contains serotonin [5-hydroxytryptamine (5-HT)]: it is not localized within either the CNS or other parts of the PNS. The distribution of serotoninergic and peptidergic (especially FMRFamide) immunostaining is widesprea d, being localized within the lateral and Parasitology Today, vol. 12, no. 9, 1996

dorsal pharyngeal nerves, the pharyngeal commissures, the nerve plexuses and associated nerve cells and fibres17(Figs 6, 7). The effects of nematode peptides ,+.~ the pharyngeal pumping behaviour of A. suum have been monitored using a pressure transducer system which measures intrapharyngeal pressure changes4°. Consistent with the localization of 5-HT-immunoreactivity to the enteric neurones and its putative transmitter role, 5-HT (10-1000 IxM) stimulates pharyngeal muscle to pump. Most interestingly, from the point of this review, this action of 5-HT is subject to peptidergic modulation. For example, the endogenous nematode FaRP, KSAYMRFamide (AFS) at nanomolar concentrations elicits a biphasic response on pharyngeal pumping behaviour4°, consisting of an initial increase in amplitude and frequency of pumping, followed by a reversible inhibition (Fig. 8)4°. The inhibition is characterized by the pharynx remaining in a hypercontracted state, with no pumping movements. This potent action of an FaRP on pharyngeal muscle strongly implicates these peptides in the control of the vital process of feeding. The central nervous system. The effects of the FaRPs on nematode central neurones have yet to be fully investigated. The only effect reported to date is that AF1 blocks the production of oscillatory potentials in inhibitory motor neuronesis. Clearly, this is an area that merits further investigation.

L

l

Inactivation mechanisms Fig. 8. Recordingshowing the effect of the nematode peptide KSAYMRFamide (AF8) A physiological role for neuropep- [50 nM (top) and 500 nM (bottom)] on pharyngeal pumping behaviour in Ascar/s. The tides in the Ascaris motor nervous bar indicates the duration of pepride application. Scale bar, horizontal = 60 s, vertical system is supported by the identifi- = 2 mm Hg. cation of a mechanism for peptidL. inactivation. AF1 is metabolized to present. The realization came initially with studies on biologically inactive fragments by an endopeptidase, the distribution of peptide immunoreactivities, but has aminopeptidase and a deamidase4L The endopeptisince been confirmed by the isolation of an everdase resembles the mammalian endopeptidase, expanding range of endogenous FaRPs. Little work has neprilysin, in that it is inhibited by phosphoramidon been done on the distribution of endogenous peptides, and thiorphan42; this observation is of particular the vast majority of immunocytochemical studies interest as a putative neprilysin gene has been identihaving utilized antisera to exogenous peptides. fied as part of the C. elegans genome sequencing However, the reaction to antisera raised to exo~,-~nproject. Therefore, analysis of the physiological conous peptides suggests that members of 7"cptide sequences of neprilysin gene knockouts in C. elegans families other than FaRPs may also be ! cesent i n will provide further information on the functional nematodes. Relatively little is known about the inacti~ importance of neuropeptide signalling systems in vation mechanisms for the putative peptide t r a ~ m i t : nematodes. Conclusions It is now well established that peptides predominate in neural signalling systems in nematodes, outstripping classical transmitters by a factor of 3:1 at Parasitology Today, vol. 12. no. 9. 1996

Reviews spp 21~,~ (C. Li, pets. commun.) and a partial cDNA sequence has been isolated from Ascaris (A.S. Edison, unpublished: NC~I GenBank Database, accession no. U15279). However, neither the latter nor the tip-1 gene encode AF2, indicating that it is likely that mor~ than one gene encodes FaRPs in nematodes. The gene sequence information will permit mapping of gene expression using in situ hybridization and facilitate the hunt for these and other peptides in other parasitic nematodes which are not so amenable to physiological study. Clearly, understanding of the biological actions of peptides is still in its infancy. The evidence so far strongly supports a role for FaRPs in the motor nervous system and in feeding behaviour; the distribution of immunoreactive neurones suggests an involvement in other processes vital to nematode survival, namely, sensory transduction and reproduction. However, in no case has the identity of the FaRP or other peptide .__ individual neurones responsible for controlling the behaviour patterns been established. Furthermore, the extent to which different nematode species share similar Peptidergic mechanisms (as they do neuronal circuitry) is not known. The nematode FaRPs are closely related structurally, yet it is clear even from the limited studies undertaken to date that relatively small changes, of as little as a single amino acid, are sufficient to alter drastically the mechanism of action induced by the ligand-receptor interaction. Further structure-activity studies will highlight the significance of specific amino acid substitutions. The complexity of intercellular signalling mechanisms (not just peptide-peptide but peptide--classical transmitter interactions) and their ionic basis remains to be evaluated. The fact that the endogenous peptides sequenced to date are FaRPs is fortuitous, in the sense that this family of peptides is of widespread occurrence throughout invertebrate phyla 43-45. Moreover, the roles of FaRPs have been investigated extensively in other well-defined invertebrate neuronal networks such as the crab stomatogastric ganglia% the Aplysia accessory radula closer muscle and in the locust motor nervous system47, for example. Consequently, there exists an excellent theoretical framework on which to base studies of these peptides in nematodes. Unlike conventional transmitters, the sequences of peptides vary between species and phyla and this makes them attractive as targets for those searching for agents tarot are selectively toxic to a parasite and not its host. Each putative peptide transmitter offers a multiplicity of targets for disruption (eg. biosynthetic pathways, pre- and post-synaptic receptors, inactivation mechanisms, ion channels, second messenger systems). Understanding of these areas will follow the sequencing of further peptides, their location to specific neuronal populations and identification of their receptors. This wilt enable the design of analogues or antagonists of peptides responsible for co-ordinating specific aspects of behaviour and make possible the realistic development of new therapeutic strategies against the many species of nematodes that are major parasites of humans, livestock and plants. References I Go!dschmldt,R. (1908)Das Nervensystemvon Ascarls lumbrlcoides und megalocephala. Z. Wiss. Zool. 90, 73-136 2 Angstadt, J.D. et al. (1989) Retrovesicular ganglion of the .... SSO

nematode Ascaris. J. Comp. Neurol. 284,374-388 3 Holden-Dye, L. and Walker, R.J. (1994) Characterization of identifiable neurones in the head ganglia of the parasitic nematode Ascaris suum: a comparisonwith central neurones of Caenorhabditis etegans. Parasitology 108, 81-87 4 White,J.G. et al. (1986) The structure of the nervous system of Caenorhabditis elegans. Philos. Trans. R. Soc. London Ser. B 314, 1-340

5 Cowden, C. et al. (1993) Localization and differential expression of FMRFamide-like immunoreactivity in the nematode Ascaris suum. ]. Comp. Neurol. 333,455--468 6 Gersch,M. and Scheffel,H. (1958)Sekretorischtiitige Zellen im Nervensystemyon Ascaris. Natumoissenschaften 45, 345-346 7 Davey,K.G.(1988)in hwertebrate Endocrinology (Vol.2) (Downer, R.G.H.and Laufer,H., eds), pp 63-86, Alan R. Liss S Brownlee,D.J.A.et aL (1993) Immunncytochemicaldemonstration of neurupeptides in the central nervous system of the roundworm,Ascaris sumn (Nematoda:Ascaroidea).Parasitolo~,/ 106, 305-316 Sithigorugul,P. et al. (1990) Neuropeptide diversity in Ascaris: an immunocytochemicalstudy. J. Comp. Neurol. 294,362-376 Brownlee, D.J.A. et al. (1994) Ultrastructural localization of pancreatic polypeptide- and FMRFamide immunoreactivities within the central nervous system of the nematode, Ascaris sumn (Nematoda:Ascarnideal. Parasitology 108,587-593 Brownlee, D.J.A. et al. (1996) Cellular and subcellular localization of SALMFamide (Sll-like immunoreactivitywithin the central nervous systemof the nematode, Ascaris sumn (Nematoda, Ascaroidea). Parasitol. Res. 82,149-156 Brownlee, D.J.A.et al. (1994) Immunocytochemicaldemonstration of neuropeptides in the peripheral nervous system of the roundworm Ascaris suum (Nematoda, Ascaruidea). Parasitol. Res. 79, 302-308 Wikgren, M. and Fagerholm, H-P. (1993) Neurupeptides in sensorystructuresof nematodes. Acta Biol. Hang. 44,133-136 Davey, K.G.(1964) Neurosecretorycells in a nematode,Ascaris lumbricoides. Can. ]. Zool. 42, 731-734 Atkinson, H.J. et al. (1988) FMRFamide-like immunoreactivity within the nervous system of the nema~.odes Panagrellus redivivus, Caenorhabditis elegans and Heterodera glyeines. J. Zool.216, 663-671 16 Schinkmann,K. and Li, C. (1992) Localization of FMRFamidelike peptides in Caenorhabditis elcgans. ]. Camp. Nearol. 316, 251-260 17 Brownlee, D.J.A. et al. (1994) Immunocytochemicaldemonstration of peptidergic and serotoninergiccomponents in the enteric nervous system of the roundworm, Ascaris suum (Nematoda, Ascaruidea). Parasitology 108, 89-103 18 Cowden,C. et al. (1989)AFI,a sequencedbioactive neuropeptide isolated from the nematode Ascarls sumn. Neuron 2,1465-1473 19 Cowden,C. and Strelton, A.O.W.(1993)AF2,an Ascaris neuropeptide: isolation, sequenceand bioactivity. PeptidL~14,423-430 20 Cowden, C. and Stretton, A.O.W. (1995) Eight novel FMRFamide-like neuropeplides isolated from the nematode Ascaris suum. Peptides 16, 4-~1-500 21 Rosoff,M.L.ct al. (1992)Alternatively spliced transcriptsof the tip-1 gene encode distinct FMRFamide-like peptides in Caenorhabditis clegans. ]. Neurosci. 12, 2356-2361 22 Rosoff,M.L.et al. (1993)The tip-1 prnpeptide is processedinto multiple, highly similar FMRFamide-!ike peptides in Caenorhabditis elegans. PeptidL~14, 331-338 23 Marks, N.J. et al. (1995) Isolation ot AF2 (KHEYLRFamide) from Caenorhabditis elegans: evidence for the pre~ance of more than one FMRFamide-related peptide-encoding gerie. Biochem. Biophys. Res. Commun. 217,8~5-851 24 Geary, T.G. et al. (1992) Two FlVlRFamide-like peptides from the free-living nematode Panagrellus redivivus. Peptides 13, 209-214 25 Maule, A.G. et al. (1994) The FMRFam~de-like neuropeptide AF2 (Ascaris sumn) is present in tb~e tree-living nematode, PanagreUus redivivus (Nematoda, Rhabditida}. Parasitology 109, 351-356 26 Maule, A.G. et aL (1994) KSAYMRFamide: a novel FMRFamide-related heptapeptide from the free.llving nematode, Panagrellus redivivus, which is myoactive in the parasitic nematode, Ascaris ~uum. Biochem. Biophys. Res. Commun. 200, 973.-980 27 Maule, A.G. et aL (1995) Isolation and preliminary biological characterization of KPNFIRFamide, a novel FMRFamiderelated peptide from the free-living nematode, Panagre!lu.; rcdlvivus. Peptides 16, 87-793 Parasitology Today, voL 12, no. 9, 1996

Reviews 28 Keating, C. et al. (1995) The FMRFamide-like neumpeptide AF2 is present in the nematode Haemonchus contort'us. Parasitology 111, 315-321 29 Santama, N. et al. (1995) Alternative RNA splicing generates diversity of neuropeptide expression in the brain of the snail Lymnaea - in-situ analysis of mutually exclusive transcripts of the FMRFamide gene. Eur. J. NeuroscL7, 65-76 30 Schinkmann, K. and Li, C. (1994) Comparison of two Caenorhabditis genes encoding FMRFamide (Phe-Met-ArgPhe-NH2)-Iike peptides. Mol. Brain Res.24, 238-246 31 Pang, F-Y. et al. (1992) The actions of acetylchotine (ACh) and a PHE-MET-ARG-PHE (FMRF)-amide-like peptide on a dorsal muscle strip of the parasitic nematode Ascaris suum. Br. J. PharmacoLI07, 458P 32 Holden-Dye, 1. et al. (1995) The neuropeptide Lys-Pro-AsnPhe-lleu-Arg-Pbeamide (l,~NFIRFamide) hyperpotarizes the somatic m':scle cc~l~ f 'he parasitic nematode Ascaris sumn. Br. J. Phanaacol. 116,, .0P 33 Pang, F-~. et al. (1995) The effects of the nematode peptide, KI'IEYLRFamide (AF2), on the somatic musculature of the parasitic nematode Ascaris suum. Parasitology110, 353-362 34 Sithigorngul, P. and Stretton, A.O.W. (1991) Differential distribution of AF1, a FMRFamide-Uke neuropeptide in Ascaris nervous system revealed by specific monoclonal antibodies. Soc. Neurosci. Abstr. 17, 279 35 Franks, C.J. et al. (1994) A nematode FMRFamide-like peptide, SDPNFLRFamide (PFI), relaxes the dorsal muscle strip preparation of Ascaris suum. Parasitologt/108, 229-236 36 Holden-Dye, L et al. (1995) The effect of the nematode peptides SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2) on synaptic transmission in the parasitic nematode Ascaris smm*.

Parasitology 110, 449-455 37 Albertson, D.G. and Thomson, J.N. (1976) The pharynx of Caenorhabditis elegans. Philos. Trans. R. Soc. :-.~ndonSet'. B 275, 299-325 38 Ward, S. et aL (1975) Electron microscopical ~onstmciion of the anterior sensory anatomy of the nematode Caenorhabditis elegans. 1. Comp.NeuroL160, 313-338 39 Brownlee, D.J.A. et al. (1995) The phagynx of the nematode Ascaris suum: structure and fur,ction. Acta Biol.Hung. 46,195-204 40 Brownlee, D.J.A. ct aL (1995) The action of serotonin and the nematode neuropeptide KSAYMRFamide on the pharyngeal muscle of the parasitic nematode, Ascar/s snare. Parasitology 111,379-384 41 Sajid, M. et aL (1996) Metabolism of AF1 (KNEHRF-NH2) in the nematode, Ascaris saum, by aminopeptidase, endopeptidase and deamidase enzymes. Mol. Biochem.Parasitol.75,159-168 42 Sajid, M. and Isaac, R.E. (1995) Identification and properties of a neuropeptide-degrading endopeptidase (neprilysin) of Ascaris suum muscle. Parasitology111,599--6~ 43 Greenberg, M.I- and Price, D.A. (1992) Relationships among the FMRFamide-like psptides. Prog. Brain Res.92, 25-37 44 Cottrell, G.A. (1989) The biology of the FMRFamide-serics of peptides in molluscs with special reference to Helix. Comp. Biochem. Physiol.93A, 41-45 45 Walker, R.J. (1992) Neuroactive l~ptides with an RFamide or Famide carboxyl terminal. Comp. Biochem.Physiol.102C, 213-222 46 Weimann, J.M. et aL (1993) The effects of SDRNFLRFamide and TRNFLRFamide on the motor patterns of the stumatogastric ganglia of the crab, Cancer borealis. ]. Exp. Biol. 181,1-26 47 Calabrese, R. L. (1989) Modulation of muscle and neummnscular junctions in invertebrates. Semh~.Neurosci. 1, 25-34

The Pharmacology of Nematode FMRFamide-related Peptides A.G. Maule, -E.G. Geary, J.W. Bowman, C. Shaw, D.V~: I2 alton and D.P. Thompson FMRFamide-related peptides (FaRPs) are ttze largest known family of invertebrate neuropeptides. Immunocytochemical screens of nematode tissues using antisera raised to these p'.ptides have localized extensive FaRP-immunostainir.g to their nervous systems. Although 21 FaRPs have been isolated and sequenced from extracts of free-living and parasitic nematodes, available cvhlence indicates that other FaRPs await discovery. While our knowledge of the pharmacology of these native nematode neuropeptides is extremely limited, reports on their physiological activity in nematodes are ever increasing. All the nematode FaRPs examined so far have been found to have potent and varied actions on nelnatode neuromuscular activity. It is only through the extensive pharmacological and physiological assessment of the" tissue, cell and receptor interactions of these peptidic messengers that an understanding of their activity on nematode neurolnlisculature will be possible. In this review, Aaron Maule and colleagttes examine the current understanding of the pharmacology of nematode FaRPs. A~'on Maule, Chris Shawand Dave Halton are at the Comparative Neuroendocrinolog,/Research Group, Schools of Biology and Biochemistry and Clinical Medicine, The Queen's University of Belfast,Belfast,UK BT7 I NN. Tim Gear'/,Jerry Bowman and Dave Thompson are at Animal Health Discover,/Research, Pharmacia and Upjohn Inc., Kalamazoo, I149001, USA. Tel: +44 1232 245133 x 2059, Fax: +44 1232 236505, e-math ~ m a u l e ~ l u b . a c . u k Parasitology Today, vol. 12, i:o. 9, 1996

T h e n e r v o u s s y s t e m of n e m a t o d e s is d i s t i n g u i s h e d b y its relatively s i m p l e c o n s t r u c t i o n ( c o m p r i s i n g 298 cells in Ascaris suum a n d 302 cells in the h e r m a p h r o d i t i c Caenorhabditis elegans) a n d b y its a r c h i t e c t u r a l similarity b e t w e e n species. This c o n f o r m i t y in n e u r o n a l c o n s t r u c tion is illustrated b y the m a j o r a n t e r i o r ganglia, w h i c h i n c l u d e the r e t r o v e s i c u l a r g a n g l i a a n d t h o s e concent r a t e d o n the d r c u m p h a r y n g e a l n e r v e ring, the l o n g i t u d i n a l ventral, d o r s a l a n d lateral n e r v e t r u n k s , a n d the p o s t e r i o r g a n g l i a , c o m m o n l y t e r m e d the c a u d a l (pertanal) ganglia. A u n i q u e a s p e c t of n e m a t o d e n e r v o u s s y s t e m s is the n a t u r e o, t h e i r n e u r o m u s c u l a r c o n n e c tivity: m u s c l e bellies s e n d o u t p r o c e s s e s (termed m u s c l e a r m s ) , w h i c h b r a n c h a n d f o r m s y n a p s e s w i t h the m a j o r v e n t r a l a n d d o r s a l n e r v e c o r d s 1 (see Fig. 1). Also, n e m a t o d e s possess o n l y l o n g i t u d i n a l s o m a t i c muscle. M o s t r e s e a r c h o n n e u r o m u s c u l a r f u n c t i o n in n e m a t o d e s h a s f o c u s e d o n A. suum, p a r t l y b e c a u s e of its size a n d c o n s e q u e n t a m e n i t y to p h y s i o l o g i c a l manipulatio,'.. T h e p i o n e e r i n g w o r k o f t h e Del Castillo a n d Str,'tcon l a b o r a t o r i e s e s t a b l i s h e d t h e f o u n d a t i o n s in o:zl c u r r e n t u n d e r s t a n d i n g o f t h e f u n c t i o n i n g of ~he n e m a t o d e n e u r o m u s c u l a r s y s t e m . M o s t o f this w o r k w a s c a r r i e d o u t p r i o r t o t h e ' p e p t i d e r e v o l u t i o n ' a n d c,o n c e n t r a t e d o n the e n d o g e n o u s classical n e u r o t r a n s m i t t e r mol~ules, k-aminobutyric acid (GABA)and acetyl choline (ACh). Evidence g e n e r a t e d l a r g e l y b y ~ la~ o r a t o r i e s r e v e a l e d the tonic release o f G A B ~ a n d A C ~ f r o m i n h i b i t o r y a n d eXcitatory n e u r o n ~ ! t ~ f i ~ l y !

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