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Organization of neuropeptidergic systems in Lymnaea

Lymnaea is a striking example of an invertebrate system where use of modern molecular biological approaches has led to a deep understanding of the role of neuropeptides in physiology and behaviour. Examples that illustrate the general principles of neuropeptide functions in the nervous system are available in this model organism. Neuropeptides are located in neural networks that control the major physiological and behavioural functions of the animal such as the cardiovascular system, reproduction, growth and metabolism and ion and water regulation (Fig. 1). Accumulating the data from molecular mapping studies on the central ganglia of the CNS (Benjamin, 2008) indicate that at least 2,500 neurons are peptidergic representing 12.5% of the estimated total CNS population of ~20,000 neurons. Taking the buccal ganglia alone a higher proportion, 25%, appears to be peptidergic. When considering the control of heavily chemically modulated peripheral target organs like the heart, gut or penis it is likely that all the controlling neurons are peptidergic. A very large number of neuropeptides are involved in controlling the muscular movements of these organs. For example, a recent peptidomic analysis of the neuropeptides involved in copulatory behaviour indicates that the penis complex contains 44 different peptides arising from 10 different genes (Filali et al., 2006).

Figure 1: Map of peptidergic neurons in the CNS of Lymnaea. The cerebral commissure is the neurohaemal organ of the CDCs. The median lip nerves (MLN) are the neurohaemal organ of the LGCs. The anterior lobe (AL) APGW neurons project along the right penis nerve (PN) and innervate the penis complex. Note that the cluster of Yellow Cells that lie on the right side of the visceral ganglion are a special group of cells whose axons project along the distal processes of the intestinal nerve to innervate the pericardium and the reno-pericardial canal (see text). The cerebral A cluster cells are an example of whole-body withdrawal response motoneurons. Abbreviations: C, cerebral ganglion; CC, Canopy Cell; B/E gp, B/E group; LL, lateral lobe; LP1, left parietal 1; LPa, left parietal ganglion; Pe, pedal ganglion; Pl, pleural ganglion; RPa, right parietal ganglion; RPD1/2, right parietal dorsal 1/2; RPeD1, right pedal dorsal 1; V, visceral ganglion; VD1, visceral dorsal 1; VD4, visceral dorsal 4; VL ventral lobe.

Hormones and transmitters

The classic separation of neuropeptides into hormones and transmitters is blurred in Lymnaea. Certainly many of the peptides are released into the blood and reach their targets via the cardiovascular system acting as neurohormones but the same peptides may act as neurotransmitters/modulators within the CNS and play a role in controlling behaviour. A good example of this is the CDCH (caudodorsal cell hormone) that acts as a hormone to stimulate ovulation but also acts as a transmitter to organize the sequence of movements that underlie egg laying behaviour. Release of peptides into the blood stream occurs at specific sites such as the nerves leaving the cerebral ganglia (Fig. 1) that act as neurohaemal organs (e.g. the CDCs, Caudodorsal Cells and the LGCs, Light Green Cells) but in other examples there are no specific neurohaemal organs and peptide release into the blood occurs more generally from nerve terminals in the connective sheath surrounding the central ganglia (e.g. the YCs, Yellow Cells and LYCs, Light Yellow Cells). However, many peptidergic neurons form synaptic connections with peripheral organs (e.g. heart or penis) or with other neurons in central circuits of the brain such as those controlling feeding behaviour. In these examples, peptides may act as primary transmitters or more frequently as co-transmitters to modulate the effects of classical transmitters such as 5-HT (serotonin) or acetylcholine (ACh).

The molecular basis of neuropeptide diversity

The neuropeptides form the largest group of direct signalling molecules in the nervous system. So far in Lymnaea 17 neuropeptide genes have been cloned and approximately 100 neuropeptides identified with certainty (examples in Table 1). This diversity in Lymnaea arises from a variety of general molecular mechanisms that occur in all types of nervous systems.

The presence of multiple gene families

A good example of this is the gene family encoding insulin related-peptides (MIPs, molluscan insulin related peptides). Five different MIP genes (MIP I, II, III, V, VII) give rise to five highly divergent MIPs (Smit et al., 1998). Another example is the CDCH gene that occurs in 3 variants (CDCH I-III) that encode related, but diverse, CDCH ovulation hormones (e.g. CDCH-1 compared with CDCH-2).

The encoding of structurally-related peptides on the same gene

This is widespread in Lymnaea where families of peptides sharing the same amino acid sequence motifs were found to be encoded on a single gene. There are examples where 2 (SCPA and SCPB, small cardioactive peptide A and B), 3 (LIPs, Lymnaea inhibitory peptides, A-C), 5 (myomodulins, LFRLamides) and 13 (F(X)RIamides) structurally-related peptides are encoded on the same gene.

The encoding of structurally-diverse peptides on the same gene

A good example is the CDCH1 gene whose single peptide precursor that encodes 10 different peptides with very diverse structures. So far 8 of these peptides have been confirmed by peptide sequencing or mass spectrometry. These are the CDCH1, the ε peptide, the δ peptide, β1 and β3 peptides, the CTP peptide, calfluxin and the α CDCP peptide (Li et al., 1994; Jemenez et al., 2004).

Alternative splicing of a primary transcript encoding different protein precursors

Alternative mRNA splicing also leads to diverse peptide expression. One of the best understood examples occurs in the gene encoding FMRFamide-related peptides (FARPS)(Benjamin & Burke, 1994; Santama & Benjamin, 2000). The single FMRFamide gene consists of 5 exons (I-V). Two different mRNA variants are spliced from the primary transcript (Fig. 2A). One (mRNA 1) consists of exons I and II the other (mRNA 2) consists of exons I, III, IV and V. Exon I encodes a highly hydrophobic sequence and an N-terminal cleavage site typical of leader sequence required for the targeting of the precursor sequences to the endoplasmic reticulum. The protein precursor 1 derived from Exon II encodes 5 different confirmed peptides FMRFamide, FLRFamide, EFLRIamide, (p)QFYRIamide and ‘SEEPLY’, a 22 amino acid peptide (Fig. 2A). The protein precursor 2 formed from the translation of exons III, IV and V encodes 7 different peptides, SDPFLRFamide, GDPFLRFamide, SDPYLRFamide, SKPYMRFamide, ‘Acidic peptide’, a 35 amino acid peptide, a ‘22 amino acid’ amidated peptide (Fig. 2A). In situ hybridization studies using either cDNA or exon-specific oligonucleotides have revealed that the two alternatively spliced mRNA species are expressed in the CNS in a striking, differential and mutually exclusive manner at the single neuron level (Fig. 2B). Of the ~ 340 neurons that express the FMRFamide gene, the majority (80%) express the mRNA 1, the rest mRNA 2 (Bright et al., 1993).

Figure 2: Alternate mRNA splicing of the FMRFamide gene. A, Two mRNA variants are spliced from the primary transcript. Protein precursor 1 encodes 5 different peptides, including multiple copies of the tetrapeptides, FMRFamide and FLRFamide. Post-translational processing of QFYRIamide converted Q into pQ. Protein precursor 2 encodes 7 peptides including multiple copies of the heptapeptides, SDPFLRFamide and GDPFLRFamide. Post-translational cleavage of the ‘acidic peptide’ resulted in two further peptides P3 and P1. Only peptides that were confirmed by sequencing and mass spectrometry are included in the list of peptides. B, In situ hybridization of the alternatively spliced transcripts shows the mutually exclusive mRNA expression at the single neuron level. The same neurons can be identified in these adjacent sections of the visceral ganglion.

Post-translational processing of peptides

Post-translational truncation of peptides has been reported for MIPs (molluscan insulin-related peptides), FARPs and the LYC peptides. Direct peptide profiling by mass spectrometry of individual LYCs has revealed a delicate pattern of peptide processing. The LYC peptide precursor is processed to yield three different peptides (LYC I, LYC II and LYC III) by cleavage at dibasic sites. LYC I and LYC II are further processed at their N-termini to yield truncated mature peptides LYCP I′ and LYCP II′ (Table 1). A recent peptidomic analysis of individual VD1/RPD2 neurons (Jimenez et al., 2006) has revealed a whole battery of different post-translational modifications including phosphorylation of an amino acid in the β peptide and a number of modified α2 modified peptides that include hydroxylated and glycosylated forms.

Peptide processing enzymes

All the sequences of peptides such as those expressed in the FMRFamide peptide precursors are flanked by mono-basic, dibasic or tetrabasic amino acids (R (arginine), RR, K(lysine) K, KR and RRKR and RKRR) that are cleavage sites for enzymes known as endoproteases. Examples of the mRNA sequences that code for the prohormone convertases (PCs) involved in endoproteolytic processing have been cloned for Lymnaea. These are LPC2 (Lymnaea PC2, Smit et al., 1992), one of a class of convertases that cleave at dibasic cleavage sites RR or KR and Lfurin1 (Lymnaea furin1) and Lfurin 2 that are known to cleave tetrabasic cleavage sites such as RKRR (Smit et al., 1994). Activation of PC2 in Lymnaea and other organisms is regulated by the molecular chaperone protein called 7B2 that inhibits the active site (Spijker et al., 1997). A final step in peptide biosynthesis is amidation of peptides by two enzymes that are synthesized by one precursor known as the α-amidating enzyme whose gene has been cloned in Lymnaea (Spijker et al., 1999a). This amidation protects peptides from degradative enzymes and is often essential for peptide bioactivity. These regulatory processes would be expected to act together in peptidergic neurons and a recent study (Spijker et al., 2004) has shown that there is neuron-specific expression of the transcripts encoding convertases, α-amidating enzyme and the 7B2 chaperone in the egg-laying hormone producing CDCs. Furthermore, environmental stimuli that induce egg laying (clean water treatment) cause co-regulated induction of the 3 transcripts and the egg laying hormone. This shows that neuropeptide release and the regulation of transcript levels of both prohormones and processing enzymes are regulated in accordance with physiological demands.

Neuropeptide Receptors

Rather less work has been carried out on the identification of the genes for neuropeptide receptors in Lymnaea and the ones that have been identified, apart from the putative molluscan insulin-related receptor (MIPR, Roovers et al., 1995), were receptors for novel peptides that were previously unknown. The single Lymnaea MIPR has the typical insulin receptor features including a cysteine rich domain, a single trans-membrane domain and a tyrosine-kinase domain. Most Lymnaea peptide receptors, like those from mammals, mediate their effects through one subgroup of GPCRs (G protein coupled receptors) that are characterized by seven alpha helical trans-membrane spanning domains and the presence of particular conserved amino acids. In Lymnaea, degenerate oligomers corresponding to these conserved amino acids were used in PCR analysis to probe cDNA libraries from the CNS or other tissues like the heart where the receptors were expected to be located. Orphan GPCR clones were expressed in Xenopus oocytes or CHO cells and putative peptide ligands from the CNS were screened for their ability to mobilize intracellular calcium. Perhaps it is not surprising that a rather random and sometimes unexpected set of receptors were discovered using this ‘shot-gun’ approach. For instance, attempts to isolate the receptors of cardioexcitatory FMRFamide family from the snail heart turned up another type of -RFamide receptor whose ligand is LyCEP (Lymnaea cardioexcitatory peptide, Table 1). This LyCEP receptor is insensitive to FMRFamide (Tensen et al., 1998). Another type of FMRFamide receptor is the FMRFamide-gated sodium channel (LsFaNaC) which is a non-GPCR type receptor containing only two trans-membrane regions (Perry et al., 2001). Other GPCRs have endogenous ligands to a leukokinin-like peptide and a 39aa neuropeptide homologous to neuropeptide Y (Table 1). An orphan GPCR, expressed widely in the CNS, is related to vertebrate galanin and nociception/orphanin-FQ receptor families (Saunders et al., 2000). From the point of view of molecular evolution, the neuropeptide receptors to the peptide lys-conopressin (Table 1) are of particular interest. A novel GPCR receptor, LSCPR1 (Lymnaea stagnalis conopressin receptor 1), mediates both the vasopressin- and oxytocin-like functions of lys-conopressin. Oxytocin-like functions are mediated by receptors located on central neurons that control the male reproductive organs and vasopressin-like functions are indicated by receptors on other neurons that modulate insulin release (van Kesteren et al., 1995). The discovery of a second lys-conopressin receptor (LSCPR2) gives a clue to the evolutionary origin of vertebrate oxytocin and a vasopressin receptors. LSCPR2 is maximally activated by both lys-conopressin and Ile-conopressin, an oxytocin-like analog of lys-conopressin, unlike LSCPR1 that only responds to lys-conopressin. Together with a study of the phylogenetic relationships of lys-conopressin receptors in Lymnaea and their vertebrate counterparts, these data suggest that LSCPR2 represents an ancestral receptor to the vasopressin/oxytocin receptor family in the vertebrates (van Kesteren et al., 1998).

Neuropeptides in action: the function of peptides in neural and endocrine networks

Heartbeat: multiple roles of neuropeptides in the control of a myogenic peripheral organ

Peptidergic control of heartbeat is the best understood example of the modulatory control of a rhythmically active muscular organ by extrinsic peptide-containing neurons. The range of general roles that peptides play as transmitters and co-transmitters is exemplified by results from the heart control system. The heart of Lymnaea is myogenic but heartbeat is controlled by a network of 5 different types of centrally located motoneurons with diverse excitatory and inhibitory effects. These neurons are all located in the visceral and right parietal ganglia with axonal projections to the heart along the pericardial branch of the intestinal nerve (Fig. 3A).

Figure 3: Heart neuropeptides. A, The location and neuropeptide content of heart motoneurons and interneurons. All 5 types of motoneurons project to the heart by axons that project along the intestinal nerve (IN). VD4 (visceral dorsal 1) and RPeD1 (right pedal dorsal 1) are interneurons that form part of the CPG that controls both cardiac and respiratory functions. B, The nerve innervation of the heart and the peptide content of the heart and pericardium. The pericardial nerve is a branch of the intestinal nerve that penetrates the pericardium to innervate both the auricle and ventricle, with more extensive branching in the auricle. Note that there are more types of peptides in the auricle than the ventricle. Cell types: Ehe, E heart excitor; Hhe, H heart excitor; Khi, K heart inhibitor; She, S heart excitor; Tpe, T pericardium.

One branch of this nerve penetrates the pericardium to innervate both regions of the heart, with the highest number of branches in the auricle (Fig. 3B). Simultaneous electrophysiological recordings of heart motoneurones and single muscle fibres in the intact heart show that they directly innervate individual heart muscle fibres (Buckett et al., 1990b,c). One of the exciting features of the investigation of the cardiac control system is that the peptides of individually dissected identified motoneurons were subjected to MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time Of Flight) spectrometric analysis as well as immunocytochemistry to firmly establish their peptide content. The same techniques were applied to the cardiac tissue itself to correlate central neuronal content with peripheral release. A total of 28 peptides have been identified in heart and pericardial tissue arising from 8 different genes indicating a rich peptidergic control of this peripheral muscular organ. The auricle is much more heavily modulated by peptides than the ventricle and of the 8 peptide families found in the heart only one, the FARP tetrapeptides (FMRFamide, FLRFamide), have been found in the ventricle (Fig. 3B).

Diverse neuropeptide transmitters encoded on the same gene have different but combinatorial effects on heartbeat

The pair of Ehe (E heart excitor) motoneurons are unique because they allow us to study the roles of the well-known cardio-excitatory peptide FMRFamide and other types of peptides that are encoded on the mRNA transcript 1 of the FARP gene (Fig. 4A). The increase frequency and amplitude produced by electrical stimulation of the Ehe cells are mimicked by the perfusion of these peptides through the heart with each type of peptide having a different detailed effect (Buckett et al., 1990a,b; Willoughby et al., 1999a,b). The absence of classical transmitters (Buckett et al., 1990b; Dobbins, 1998) suggests that these gene-related peptides are primary transmitters mediating the monosynaptic effects that the Ehe cells have on heart muscle fibres. The peptides processed from the prohormone precursor 1 are divided into 3 types. In addition to the RFamides (FMRFamide, FLRFamide) there are the RI amides (pQFYRIamide, EFLRIamide) and the 22 amino peptide ‘SEEPLY’ (Table 1). All five peptides are found in the Ehe neurons (Fig. 4Ai), the pericardial nerve and the auricle. RIA (radioimmunoassay) analysis of heart perfusates following heart electrical stimulation shows that all three peptide types are released from the heart tissue in a Ca++-dependent manner (Dobbins, 1998). The RI amide and RFamide peptides are released in about equal quantities suggesting co-release and this was confirmed by double immuno-gold staining of secretory granules in nerve terminals in the heart. This showed that both types of peptides are present in the same type of secretory granule (Dobbins, 1998). SEEPLY appear to be released separately from different granules, and in less quantities, but is still part of the same cocktail of peptides shown to be released by heart electrical stimulation. The three types of peptides have distinct effects on heartbeat and these are mediated by different second messenger pathways. The increases in the frequency and amplitude of heart-beat produced by FMRFamide/FLRFamide (Fig. 4Aii) are mediated by mobilising the inositol phosphate pathway and they follow the same time course (Willoughby et al., 1999a). The effects of the RIamide peptides on heart beat are mediated by a separate cyclic AMP-mediated pathway that produces a more prolonged excitatory effects on heartbeat compared to the RFamides (Fig. 4 Aiii) (Willoughby et al., 1999b). An initial cyclic AMP-independent inhibitory response on heartbeat to one of the RIamides, EFLRIamide (Fig. 4Aiii), is due to direct effects on heart muscle ion channels. SEEPLY has no effects on heart-beat when applied alone but prolongs the effects of FMRFamide by delaying its mobilizing effects on the inisotol phosphate pathway. At the level of ion channels, patch clamp experiments on isolated ventricular muscle cells show that gating effects of FMRFamide on non-voltage gated Ca2+ channels is prolonged by the co-application of SEEPLY to the outside of the muscle fibre (Brezden et al., 1999). In the absence of SEEPLY, repeated application of FMRFamide leads to a progressive reduction in the opening frequency of the Ca2+ channels. We conclude that different types of receptor-mediated molecular and ionic mechanisms underlie the effects of FMRFamide, RIamide and SEEPLY. The RIamides prolong the excitatory effects of FMRFamide, with early inhibitory effects acting to maintain a steady increase in beat rate. SEEPLY is modulatory helping to prevent the a loss in responses to repeated application of FMRFamide. Their co-release from the Ehe motoneurons means that their individual effects are integrated to control heart function.

Figure 4: Synaptic function of identified neurons expressing precursor 1 and precursor 2 peptides of the FMRFamide family. Ai, MALDI-TOF analysis of a dissected Ehe heart excitor motoneuron shows the presence of precursor 1 peptides. Aii, Perfusion of FMRFamide through the heart increases beat rate and amplitude as does bursts of induced spikes in the Ehe cell. Neuronal bursts produce longer duration effects than peptide application as shown in the plots of instantaneous beat rate (bpm, beats per minute) in the upper trace. Aiii, EFLFIamide initially inhibits spontaneous heartbeat and then increases beat rate and amplitude, similar to FMRFamide, but over a more prolonged time period. Bi, VD4 only expresses precursor 2 peptides. No precursor 1 peptides are present. Bii, An evoked burst of spikes in VD4 generates a biphasic depolarizing (e) followed by hyperpolarising synaptic response (i) in RPeD1 (right pedal D 1). Its persistence in high Mg2+/high Ca2+ saline indicates a monosynaptic connection. Biii, Application of SDPFLRFamide/GDPFLRFamide produces a hyperpolarising response, ACh a depolarising response (Biv), and a mixture of both produces a biphasic response (Bv) similar to neuronal stimulation.

Diverse neuropeptide transmitters encoded on different genes converge on the same heart ion channel

VD1/RPeD2 (Fig. 3A) are functionally-related giant neurons that control a number of physiological processes related to pO2 regulation that includes the modulation of heartbeat. They fire as a single unit in a 1:1 pattern due to their strong electrotonic coupling (Benjamin & Pilkington, 1986). VD1/RPeD2 express a wide range of neuropeptides that are encoded on multiple genes. Both neurons express the α1 and α2 gene-related peptides (Table 1) and a novel 53 amino acid LyCCAP (Lymnaea calcium current activating peptide) that is encoded on a different gene. VD1 alone expresses the two SCP peptides (Table 1) encoded on a third gene. Despite the structural diversity of these peptides they all target the same ion channel. When synthetic versions of the α2, LyCCAP and SCPA and SCPB peptides are applied to isolated ventricular cells they all specifically enhance the size of the HVA (high voltage activated) L type Ca2+ current, although with varying potencies (Jimenez et al., 2006). It is interesting that post-translationally-modified mono and di-glycosylated forms of the α2 peptide are more potent than the unmodified isoform. These four types of peptides appear to be the only transmitters present that mediate the excitatory synaptic effects that VD1 makes with the isolated ventricular cells in culture. The HVA Ca2+ currents in heart ventricular cells are important in generating the pacemaker mechanism of the myogenic heartbeat (Yeoman & Benjamin, 1999) so one suggested function for the VD1/RPeD2 peptides is to increase the mygogenic beating rate.

Neuropeptides act as co-transmitters in heart motoneurons

5-HT is the classical transmitter of two types of excitatory heart motoneurons, the Hhe (H heart excitor) and the She (S heart heart excitor) cells. The main excitatory effects that these neurons have on heartbeat is mediated by this monoamine (Buckett et al., 1999c). However, one of these neurons, the Hhe, expresses remarkably a large number of peptide co-transmitters that modulate the effects of 5-HT (Dobbins, 1998). The Hhe cell body has been shown to contain members of 4 different peptide families, the myomodulins, SCPA,B, LIPs and α1/α2 (Fig. 3A) with 12 different peptides identified by MALDI-TOF spectrometry of dissected neurons (Worster 1996; Dobbins, 1998). All these peptides have also been found in the auricle (Fig. 3B). The SCPs have highly potent effects on the auricle (threshold 10-10 M), and similar to 5-HT act to increase beat rate but in a more prolonged manner. Importantly, these peptides also increase heart tonus. This increase in tonus is observed with neuronal stimulation but is not mimicked by application of 5-HT to the heart suggesting that it is one of the specific modulatory roles of SCP. The effects of mymodulin have not been tested on the Lymnaea heart, but in the nudibranch mollusc Archidoris, mymodulins act as modulators to potentiate the cardioexcitatory effects of 5-HT, preventing desensitization effects that occur following repeated application of the monoamine (Wiens and Brownell, 1995). This is also likely to be important in Lymnaea because repeated electrical activation of Hhe neurons does not result in a reduction in heartbeat. The α peptides increase the cellular excitability of heart muscle fibres and increase beating as previously explained for the VD1/RPD1 neurons (see above). Thus the peptide co-transmitters in the Hhe cells modulate the effects of 5-HT and in combination mediate the effects of neuronal stimulation. In other examples, the Khi (K heart inhibitor) and Tpe (T heart pericardium), ACh is the classical inhibitory transmitter on heartbeat (Buckett et al., 1990a). Here peptide co-transmitters, extended peptides of the FARP family e.g. SDPFLRamide and GDPFLRamide found in the Tpe motoneuron underlie its ability to increase cardiac tonus.

Interneuronal control of heart peptidergic motoneurons

Several types of interneurons control spike activity in peptidergic heart motoneurons. Two of these form part of the CPG for rhythmic pneumostome movements and so respiration and heart-beat are linked by a common control mechanism (see Benjamin 2008). The most significant effect of the CPG arises from excitatory effects on the Hhe motoneurons. This generates periodic bursts of spikes that excite the heart in a cyclical manner. At the same time the inhibitory input to the heart provided by the Khi motoneurons is suppressed promoting further excitation to the heart. An important interneuron in the respiratory/heart CPG is VD4. ACh and FARP peptides of the extended heptapeptide type (SDPFLRFamide/GDP etc.) are co-transmitters in this neuron (Fig. 4Bi) (Skingsley et al., 1993; Staddon, 1996; Worster et al., 1998). Spikes in VD4 generate biphasic excitatory/inhibitory monosynaptic responses on another CPG interneuron, RPeD1 (Fig. 4Bii). ACh mediates an early nicotinic depolarizing response on RPeD1 (Fig. 4Biv) and the extended RFamides a delayed hyperpolarizing response (Fig. 4Biii). Applied together they mimic the synaptic effects of VD4 stimulation (Fig. 4Bv). This is an important example of a neuron that uses peptides processed from mRNA 2 variant of the FMRFamide gene as co-transmitters and allows direct comparison with the Ehe motoneurons that express the alternative mRNA 1 (Fig. 4A).

Feeding: role of neuropeptides as co-transmitters in the modulation of a complex central motor network

Feeding in Lymnaea is an example of a more complex CNS network, a central pattern generator (CPG) interneuronal network that generates rhythmic feeding movements (Benjamin, 2008). There are about 100 neurons known to be involved in generating feeding movements and their functions can be conveniently categorized into motoneurons, CPG interneurons and modulatory interneurons (Benjamin, 2012). These neurons are mainly located in the buccal ganglia but there are a number of interneurons in the cerebral ganglia that are functionally linked to the buccal feeding network (Fig. 5). All three of these neuron types contain neuropeptides as do their target organs the oesophagus and the buccal mass (Fig. 5). A total of 47 different peptides have been identified in the buccal ganglia arising from 12 different genes. Initial immunocytochemical studies (Santama et al., 2004) revealed the widespread presence of peptides of the SCP and myomodulin families. Later, the presence of these and other peptides were confirmed using more specific probes provided by information derived from gene cloning and peptide sequencing studies (Fig. 5, Table 1). In the example of larger motoneurons such as B1 and B2 and modulatory interneurons such as the CGCs (cerebral giant cells), the ability to dissect individually identified neurons and their nerve projections for MALDI-TOF analysis was particularly valuable.

Motoneurons and peripheral target organs

The largest of the paired buccal neurons B1 (salivary gland motoneuron) and B2 (oesophageal motoneuron) both contain SCPA and SCPB and a variety of the myomodulin family peptides (Fig. 5) (Kellett et al., 1996) as well as the classical transmitter ACh (Perry et al., 1998). The isolated oesophagus is capable of spontaneous rhythmic contractions but its contractions are modulated by electrical activity in the B2 neurons (Perry et al., 1998). The frequency, amplitude and underlying tonus of these contractions are increased by B2 stimulation. The peptides and ACh each have distinct effects on the oesophagus when applied separately but act co-operatively to mimic the effect of B2 stimulation. ACh increases the frequency and amplitude of oesophageal contractions. Myomodulins have similar effects to ACh but are slower in onset compared with ACh and involve a different signalling pathway. Both SCP peptides (A and B) increase the underlying tonus of oesophageal contractions to mimic another effect of B2 stimulation (Perry et al., 1999). There are other peptides in the pro-oesophagus that originate from unidentified motoneurons on the buccal ganglia. For example, the Lymnaea tetradecapeptide (Table 1) (Li et al., 1993). This peptide has excitatory effects on the isolated oesophagus. Other types of large buccal motoneurons, the paired B4/B8s and the B4CL cells (up to six on each side) innervate buccal mass muscles that are directly responsible for the swallow and rasp phases, respectively, of the feeding ingestive cycle. The rasp phase of the feeding cycles appears to be modulated more than the swallow phase because the B4CL neurons contain myomodulin and SCP peptides but no peptides have so far been found in the B4s. The rasp phase of feeding is under more dynamic modulatory control because the strength of the bite varies according to the ‘hardness’ of the food substrate.

Central pattern generator interneurons

The N1, N2 and N3 CPG interneurons fire in sequence to generate the protraction, rasp and swallow phases of the rhythmic feeding cycle, respectively. Immunostaining of an electrophysiologically-characterized N1M indicates the presence of a buccalin-like peptide (Santama et al., 1994). The N1M is first type of neuron in feeding system of Lymnaea that has been shown to contain this type of peptide, although work on Aplysia indicates that it is likely to have an important role in the feeding system. The N2v has been shown to express the myomodulin and SCP peptides (Fig. 5). Synaptic modulation by peptides would be appropriate in rasp, the most flexible phase of the feeding cycle. None of the N3 cells have been shown to contain neuropeptides (Santama et al., 1994). Classical transmitters mediate the main synaptic effects of CPG interneurons, ACh in the N1Ms and glutamate in the N2s and so the peptides act as co-transmitters.

Figure 5: Neuropeptide expression in neurons of the feeding system. Peptide-containing neurons are colour-coded according to function. Motoneurons are yellow, CPG interneurons are red and modulatory interneurons are dark blue. Abbreviations: CBC, cerebrobuccal connective; CV1, cerebroventral 1; DBN, dorsobuccal nerve; LBN, laterobuccal nerve; MLN, median lip nerve; NO, nitric oxide; SLN, superior lip nerve: VBN, ventrobuccal nerve. See the text for description of neuron types.

Modulatory interneurons

The well-characterized CGCs and SO (slow oscillator) interneurons have been analysed for their peptide content. Both express the myomodulin gene and its processed peptides (Fig. 5). In addition the SO expresses the Lymnaea version of the Aplysia pedal peptide. This was unexpected as the work in Aplysia suggested that the homologous peptide was involve in control of pedal locomotion (Hall and Lloyd, 1990). 5-HT is the classical transmitter of the CGCs and ACh is the classical transmitter of the SO. A cerebrobuccal interneuron (CBI), the CBWC (cerebrobuccal white cell), has mixed excitatory and inhibitory effects on the feeding circuit that are mediated by the peptide APGW (McCrohan & Croll, 1997). Application of APGW mimics the effects of neuronal stimulation to initiate feeding rhythms in a quiescent preparation but disrupts on-going rhythms, indicating a dual excitatory-inhibitory role in feeding. There is another peptidergic modulatory interneuron, the pleural-buccal interneuron (PlB), whose paired cell bodies lie outside the feeding circuit (Alania et al., 2004). It has inhibitory synaptic connections with interneurons and motoneurons of the feeding circuit and stimulating PlB suppresses feeding motor patterns. The PlB has been shown to contain exon II peptides of the FMRFamide family and applying FMRFamide to the buccal ganglia mimics the inhibitory effects of neuronal stimulation on feeding. Thus although FMRFamide is not significant as an intrinsic signalling molecule within the feeding circuit it has an important extrinsic modulatory role in controlling feeding behaviour.

Female and male reproductive behaviour: neuropeptides act as both hormones and neuromodulators in generating complex behavioural sequences

The role of neuropeptides in reproductive behaviour has been extensively investigated (Koene, 2010). Egg laying behaviour in Lymnaea and Aplysia is considered to be a prime example of a complex behaviour programmed by the central release of multiple peptides encoded by a small family of genes. Egg laying is triggered by synchronous electrical firing in several hundred caudodorsal cells (CDCs) located in the cerebral ganglia of Lymnaea (Fig. 1). The CDCs express 3 different CDCH genes that give rise to at least 10 different peptides. The best understood of these peptides is the caudodorsal cell hormone (CDCH-1, Table 1), the ovulation hormone. Release of this hormone into the blood follows CDC firing and when CDCH is injected into animals it evokes ovulation, egg mass formation and oviposition. CDCH and other peptides encoded on the CDCH gene have a variety of roles in controlling the neural circuitry associated with the different phases of egg laying behaviour (Benjamin, 2008). For example, pedal ganglion motoneurons involved in turning behaviour are excited by the β3 CDC peptide but are inhibited by the ovulation hormone because turning is suppressed during oviposition. Injection of β3 and αCDC peptides into intact snails increase the rate of rasping movements of the radula that occur during the turning phase of the natural behaviour. The rasping movements clean the substrate to allow the subsequent deposition of the egg mass. This is a distinct behaviour from the normal role of rasping in food ingestion and is accompanied by changes in the firing pattern of motoneurons and the modulatory CGCs that are ‘re-programmed’ for their role in egg laying (Jansen et al., 1997, 1999). The CDCH and the αCDC peptide together also play an interesting role as autotransmitters in the induction of the long-lasting spike discharge of the CDC cells that accompanies egg laying. Combined application of these two peptides triggers sustained firing of the cells. Generation of after-discharges by electrical stimulation is prevented by application of antibodies to the CDCH or the α CDC peptide showing they are necessary for the maintenance of firing in the CDCs (Brussard et al., 1990). Thus the release of these two specific peptides provides a self-sustaining mechanism for maintaining firing of the CDC neurons.

In male behaviour, the emphasis has been on the role of peptides in the control of penis movements. The penis is innervated by a single penis nerve originating from the right cerebral ganglia (Fig. 1). Neurons that are located in the anterior lobe (AL) of this ganglion are known to co-express a variety of peptides encoded by 4 neuropeptide genes (de Lange et al., 1997). They provide peptidergic motor innervation to muscles of the penis complex that includes the preputium, a muscular structure that surrounds the penis. Artificial electrical stimulation of the AL neurons results in the eversion of the preputium, the first stage in a sequence of movements leading to copulation. This behaviour results from the relaxation of the penis retractor muscles. APGW, originating from the anterior lobe neurons (Fig. 1), is the most important peptide mediating these muscle relaxations. APGW inhibits contraction of the penis retractor muscles in vitro and injecting APGW into the intact animal reliably evokes preputial eversion. Lys-conopressin is often co-localised with APGW in the AL neurons and significantly its stimulatory effects on retractor muscles is opposed by APGW. In other AL neurons, APGW is co-expressed with LyNPY (Table 1) and in this type of AL lobe neuron both peptides act together as synergistic co-transmitters to relax retractor muscles. There are a number of other peptides present in the penial complex (e.g. myomodulins and LIPs) that have the ability to relax retractor muscles and these could also be involved in preputial eversion. The vas deferens, which transports semen to the penis by rhythmic contractions, is another target for peptides of the male reproductive system. Lys-conopressin increases this contractile activity antagonistically to APGW. Members of another large family of peptides present in the penis complex, the F(X)RIamides (Table 1), inhibit spontaneous vas deferens contractions and could be involved in suppressing male reproductive activity.

The hermaphroditic lifestyle of Lymnaea requires that the female and male functions should not be performed simultaneously. There is good evidence that APGW also plays a key role in female suppression. Application of APGW hyperpolarizes the CDCs and prevents their after-discharge. A single interneuron, the RN (ring neuron) contains APGW (Croll & van Minnen, 1992) and is likely to provide the neural pathway responsible for the inhibitory effect of the male reproductive system on the CDCs (Jansen et al., 1985). Other peptides such as lys-conopressin and FMRFamide also inhibit CDC discharges and they could also be involved in female suppression.

Ion and water regulation: hormonal control of ion transport

Lymnaea is a freshwater snail that efficiently maintains its body fluids at a higher osmolarity than the surrounding environment by the uptake of ions from the outside medium and the excretion of a dilute urine from the kidney. An important part of the mechanism that underlies the maintenance of this hyper-osmolarity is the active uptake of Na+ ions from the outside medium to the haemolymph by an integumental Na+ pump. This pump is stimulated by the 77 amino acid peptide, SIS (sodium stimulating hormone) whose primary structure (Table 1) was determined by peptide sequencing (de Witt et al., 1993) and confirmed by cloning of the single cDNA that encodes the peptide prohormone (Smit et al., 1993). This peptide is proposed to be part of a negative feedback system for the control of blood osmolarity. The role of SIS in stimulating Na+ uptake was initially demonstrated in the intact snail by injecting the extracts of SIS-containing neurons into the haemolymph (de Witt & van der Schors, 1986). Specific effects on skin transport were later demonstrated in elegant experiments using an in vitro ‘stripped’ skin bioassay. SIS increased the size of the electrical potential difference and a short circuit current across the skin and ouabain application blocked these currents, indicating that Na+ ion transport was being stimulated (de Witt et al., 1988). Immunocytochemistry and in situ hybridization showed that the SIS peptide is expressed in the central peptidergic neurons called the Yellow Cells (YCs) (Boer et al., 1992). Many of these scattered cells appear to release SIS hormonally into the blood from fine neuritic branches that penetrate the vascular connective tissue surrounding the central ganglia to target the skin. However, there is a special identified group of YCs in the visceral ganglia (Swindale & Benjamin, 1976, Fig. 1) whose axons project along the distal processes of the intestinal nerve to innervate a number of peripheral organs that include the pericardium of the heart and kidney ducts like the reno-pericardial canal (Fig. 3B) and the ureter (Boer et al., 1992). This indicates that peripheral targeting of the SIS peptide is likely to be involved in stimulating renal uptake of Na+ ions from the pro-urine that is produced by ultra-filtration of the blood across the auricular walls to the pericardial cavity (Fig. 3B). This conclusion was supported by earlier electron microscopic studies that showed peripheral release of peptides from YC terminals in these organs was increased when snails were subjected to a hypo-osmotic environment (Wendelaar Bonga, 1972). Thus the SIS peptide acts to maintain blood Na+ concentration by stimulating Na+ uptake across the skin but also by reducing the loss of Na+ ions in the urine by peripheral reabsorption of Na+ in the cardiac-renal system. Other targeting of YC nerve fibres on the muscle fibres of peripheral organs including blood vessels suggest that the SIS peptide may be involved in controlling blood pressure as well as haemolymph ion concentration suggesting a more comprehensive role in ion and water regulation.

Growth and metabolism: hormonal role of insulin-related peptides

Extensive endocrinological experiments using the classical methods of extirpation and transplantation showed that the Light Green Cells (LGCs) are involved in the hormonal control of body growth and metabolic processes related to growth such as breakdown of glycogen and stimulation of shell growth (Geraerts, 1976). About 100 LGC neurons lie in each cerebral ganglion and their axons project to the ipsilateral medial lip nerve that is the site of release of peptides into the blood (Fig. 1). The LGCs exert their growth functions by producing and releasing insulin-related peptides (MIPs) that are encoded by a family of 5 related genes (MIP genes) (Smit et al., 1998). cDNA cloning and peptide characterization demonstrate that the LGCs express five structurally diverse peptides, MIP I-II, V-VII. Each MIP prohormone consists of A, B and C domains separated by dibasic cleavage sites. Processing results in mature insulin related peptides (A and B chains connected by disulphide bridges) and C-chains. The MIPs are structurally diverse although they share the cysteine backbone with vertebrate insulins. The structures of Lymnaea C peptides are more conserved. Although the population of LGCs express all five of the functional MIP genes (MIP IV and VI are pseudogenes) individual LGCS appear to contain only subsets of the gene products. This suggests that there may be functional differentiation of the MIP peptides. This is supported by experiments showing that food deprivation decreases the expression of the MIP genes II and V in the LGCs whereas the expression of the other genes was unaffected (Geraerts et al., 1991). Differential functions for the MIP peptides might be thought to require different receptors but Southern Blot analysis and extensive screening of cDNA and genomic clones led to the discovery of only a single MIP receptor gene suggesting that the 5 different MIPs may exert their function by binding to the same receptor. However, there may be other types of MIP receptors such as those related to tyrosine kinase receptors that have structural relationships to insulin-related receptors. Although the regulation of growth by the LGCs appears to require the MIPs derived from all 5 MIP genes there is an ectopic population of neurons in the buccal ganglia (Fig. 5) that only express the MIP VII gene (Smit et al., 1996). This suggests that the MIPs encoded on this gene have a special role in the control of feeding (Geraerts, 1976a).

It is not known for certain what physiological factors stimulate the release of MIP release but good evidence suggests that lys-conopressin could be part of the mechanism. The receptor for lys-conopressin is expressed in the LGCs and application of the peptide drives spiking activity in isolated LGCs (van Kesteren et al., 1995). This is significant because spike activity in the LGCs causes the release of MIPs into the blood. There also is an inhibitory mechanism on the LGCs that is mediated by another type of ectopic MIP expressing neuron, the Canopy Cell (CC), that is located in the lateral lobe (LL), a small extension of the cerebral ganglion (Fig. 1). Cauterization of the LL results in giant growth due to the removal of this inhibitory control (Geraerts, 1976b). Further types of hormonal effects of the LL mediate interactions between growth regulation and reproduction. Egg laying and growth both demand high levels of metabolites and energy and this makes the two processes potentially antagonistic. Removal of the LL as well as stimulating growth reduces female reproduction so the LL is thought to be the coordinating centre for this interaction between the two competing processes.

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Lymnaea neuropeptide genes

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