The neuronal basis of feeding in the snail, Helisoma, with ... · Progress in Neurobiology 63 (2001) 383–408 The neuronal basis of feeding in the snail, Helisoma, with comparisons

Progress in Neurobiology 63 (2001) 383–408

The neuronal basis of feeding in the snail, Helisoma, withcomparisons to selected gastropods

A. Don MurphyDepartment of Biological Sciences and Laboratory of Integrati6e Neuroscience, Uni6ersity of Illinois at Chicago, Chicago, IL 60607, USA

Abstract

Research on identified neurons during the last quarter century was forecast at a conference in 1973 that discussed ‘neuronalmechanisms of coordination in simple systems.’ The focus of the conference was on the neuronal control of simple stereotypedbehavioral acts. Participants discussing the future of such research called for a comparative approach; emphasis on structure–function interactions; attention to environmental and behavioral context; and the development of new techniques. Significantly,in some cases amazing progress has been made in these areas. Major conclusions of the last quarter century are that so-calledsimple behaviors and the neural circuitry underlying them tend to be less simple, more flexible, and more highly modulated thanoriginally imagined. However, the comparative approach has, as yet, failed to reach its potential. Molluscan preparations, alongwith arthropods and annelids, have always been at the forefront of neuroethological studies. Circuitry underlying feeding has beenstudied in a handful of species of gastropod molluscs. These studies have contributed substantially to our understanding ofsensorimotor organization, the hierarchical control of behavior and coordination of multiple behaviors, and the organization andmodulation of central pattern generators. However, direct interspecific comparisons of feeding circuitry and potentiallyhomologous neurons have been lacking. This is unfortunate because much of the vast radiation of the class Gastropoda isassociated with variations in feeding behaviors and feeding apparatuses, providing ample substrates for comparative studiesincluding the evolution of defined circuitry. Here, the neural organization of feeding in the snail, Helisoma, is examined critically.Possible direct interspecific comparisons of neural circuitry and potentially homologous neurons are made. A universal model forcentral pattern generators underlying rasping feeding is proposed. Future comparative studies can be expected to combinebehavioral, morphological, electrophysiological, molecular and genetic techniques to identify neurons and define neural circuitry.Digital resources will undoubtedly be exploited to organize and interface databases allowing illumination of the evolution ofhomologous identified neurons and defined neural circuitry in the context of behavioral change. © 2001 Elsevier Science Ltd. Allrights reserved.

Keywords: Aplysia; Heterobuccal nerve; Ventrobuccal nerve; Postsynaptic nerve

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

2. Investigations of oral behaviors and underlying neural mechanisms in gastropod molluscs . . . . 385

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Abbre6iations: A, Aplysia ; aj, anterior jugalis; B, buccal; BC, buccal commisure; BCN1, buccocerebral neuron, type 1; C, Clione ; CBC,cerebrobuccal connective; con, Contralateral; CPG, central pattern generator; DBN, dorsobuccal nerve; E, excitatory; EPSP, excitatorypostsynaptic potential; ET, esophageal trunk; GN, gastric nerve; H, Helisoma ; HBN, Heterobuccal nerve; I, inhibitory; in, influential; Ip,ipsilateral; IPSP, inhibitory postsynaptic potential; L, Lymnaea ; LBN, laterobuccal nerve; MN, motor neuron; nor, nitric oxide responsive; P,Planorbis ; PBN, posterobuccal nerve; pj, posterior jugalis; PSP, postsynaptic potential; rad tens, radular tensor; RN, radular nerve; S1, subunit1; S2, subunit 2; S3, subunit 3; SN, salivary nerve; slrt, supralateral radular tensor; SRT, subradular tissue; VBN, ventrobuccal nerve.

E-mail address: [email protected] (A.D. Murphy).

0301-0082/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 3 0 1 -0082 (00 )00049 -6

A.D. Murphy / Progress in Neurobiology 63 (2001) 383–408384

2.1. Helisoma tri6ol6is as a neuroethological model system . . . . . . . . . . . . . . . . . . . . . 3852.2. Feeding behavior and its anatomical basis in basommatophoran snails. . . . . . . . . . . . 385

3. Neurophysiological correlates of feeding in Helisoma. . . . . . . . . . . . . . . . . . . . . . . . . . 3863.1. The original model of the Helisoma buccal CPG . . . . . . . . . . . . . . . . . . . . . . . . 3883.2. A triphasic buccal motor pattern underlies feeding behavior . . . . . . . . . . . . . . . . . . 3883.3. The current model of the multifunctional buccal CPG . . . . . . . . . . . . . . . . . . . . . 3903.4. Plasticity of organization in a multifunctional CPG . . . . . . . . . . . . . . . . . . . . . . . 3913.5. Identified CPG interneurons and motor neurons. . . . . . . . . . . . . . . . . . . . . . . . . 3913.6. Evidence that the triphasic standard buccal motor pattern mediates the typical feeding

behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3923.7. Regurgitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3933.8. Transmitters of CPG interneurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3943.9. Sensory and modulatory pathways regulating the buccal CPG. . . . . . . . . . . . . . . . . 3953.10. Higher order modulation of the CPG. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

4. Comparisons of gastropod neurons and feeding circuitry . . . . . . . . . . . . . . . . . . . . . . . 3964.1. Biological and methodological complications for interspecific comparisons of gastropod

feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3964.1.1. Incomplete data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3964.1.2. Interspecific diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3974.1.3. Intraspecific diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3974.1.4. Non-congruence of interneuronal and motor neuronal patterns . . . . . . . . . . . 398

4.2. Specific buccal motor patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3984.3. Interspecific comparisons of CPG interneurons and feeding motor neurons . . . . . . . . . 3994.4. Protraction phase interneurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4014.5. Motor and influential neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4024.6. Retraction phase interneurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4024.7. Hyper-retraction phase interneurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403

5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

1. Introduction

There was a remarkable symposium, titled ‘Neuroidand Neuronal mechanisms of Coordination in SimpleSystems’, held at the annual meeting of the AmericanSociety of Zoologists in December, 1973 (Kammer andWestfall, 1974). The symposium summarized the stateof research, a quarter century ago, on mechanisms ofcoordination in a number of systems of identifiableneurons as well as in non-neural systems. The focus wason the neuronal (and non-neuronal) control of simple‘stereotyped’ behavioral acts, or, in the ethological lan-guage of the time, the neural bases underlying ‘fixedaction patterns’ (e.g. Kater, 1974; Kristan, 1974; Lent,1974; Mayeri et al., 1974; Naitoh, 1974; Selverston,1974; Spencer, 1974; Stein, 1974). Hierarchical interac-tions among potential behaviors and the modulation ofbehavior by learning were addressed (Davis et al.,1974). Participants discussed the future of such re-search. They called for, a comparative approach; em-phasis on structural and functional interactions;

attention to the environmental and behavioral contextin which the experimental subject normally functions;and the development of new techniques. In addition,the marriage of genetics and neurobiology was presagedat the conference (Hoy, 1974; Ikeda and Kaplan, 1974;Levinthal, 1974), though the recent explosion of molec-ular biology and its effects on neurobiology could notbe anticipated.

In the ensuing quarter century, significant progresshas been, and continues, to be made in each of theseareas. A consensus has arisen that the behavioral out-puts of such simple systems is not so stereotyped as firstthought and that even simple neural circuitry is highlymodulated (e.g. Harris-Warrick and Marder, 1991;Marder and Calabrese, 1996). Progress in neurogeneticshas exploded. New staining and recording techniquesand application of molecular biology techniques haveilluminated structure–function relationships at levelsunimagined in 1973. Perhaps the area of inquiry pro-posed at the symposium 25 years ago that has fulfilledits potential least is the emphasis on comparative stud-

A.D. Murphy / Progress in Neurobiology 63 (2001) 383–408 385

ies and, in particular, what such studies might tell usabout the evolution of neural systems and behaviors. Iwill suggest below that the potential for such studieshas not decreased and that the comparative study of theneurobiology of molluscan feeding is pregnant withpossibilities.

2. Investigations of oral behaviors and underlyingneural mechanisms in gastropod molluscs

Investigations of the neuronal control of feeding inseveral different opisthobranch and pulmonate gas-tropod molluscs began in earnest in the early 1970s (e.g.Levitan et al., 1970; Gardner, 1971; Rose, 1971; Berry,1972; Kater, 1974; Kupfermann, 1974; Siegler et al.,1974). Opisthobranchs and pulmonates have a numberof large neurons that are identifiable uniquely in eachindividual of a species. These uniquely identifiable neu-rons have facilitated greatly the physiological andanatomical circuit analyses.

In addition, the presence of uniquely identifiableneurons indicates heritable developmental programs thatgenerate these neurons, thus offering the possibility foranalyses of the adaptive radiation of feeding circuitry.Though the potential for comparative studies of gas-tropod feeding circuitry to illuminate aspects of theevolution of homologous neurons and neural circuits wasrecognized in the early 1970s (cf. last paragraph of Kater,1974), that potential remains largely unrealized. Feedingbehaviors and their neural bases have now been more orless studied intensively in several gastropod species suchas Aplysia, Clione, Helix, Helisoma, Incilaria, Limax,Na6anax, Planorbis, Planorbarius, Pleurobranchea andTritonia (Bulloch and Dorsett, 1979; Brace and Quicke,1981; Cohan and Mpitsos, 1983; Peters and Altrup, 1984;Arshavsky et al., 1988a,b,c, 1989; Cappell et al., 1989a,b;Delaney and Gelperin, 1990; Kobatake et al., 1992;Church and Lloyd, 1994; Quinlan et al., 1995, 1997).Surprisingly, however, little effort has been made toexamine the similarities and differences in the neuralorganization of feeding in the various gastropods.

This report will focus on the neural bases of feedingand regurgitation in the snail, Helisoma tri6ol6is (Mol-lusca, Gastropoda, Pulmonata, Basommatophora,Planorbidae). It also proposes the working hypothesisthat the similarities outweigh greatly the differences inthe neural organization of feeding in those well-studiedgrazing gastropods that feed with rasping or graspingradulae (e.g. the opisthobranchs Aplysia, and Tritonia,the basommatophoran snails, Helisoma, Planorbis,Planorbarius, and Lymnaea and stylomatophoran snailsand slugs, Helix, Limax,). A model developed to repre-sent the organization of a multifunctional buccal centralpattern generator (CPG) in Helisoma is offered as ageneral model for gastropod buccal CPGs. No generally

applicable neural organization underlying gastropodfeeding behaviors has been proposed previously. TheHelisoma model will provide a reference point for exam-ining the adaptive radiation of buccal circuitry. Thesegastropod buccal CPGs appear to be comprised ofmultiple interneuronal subunits, much like the unit CPGshypothesized to control joint movements during verte-brate locomotion (cf. Grillner and Wallen, 1985). Theindividual CPG subunits are conditional neuronal oscil-lators, which can be independently activated. The activ-ities of CPG subunits too can be linked functionally indifferent combinations and in different temporal se-quences to produce different behaviors. Both physiolog-ical state and acute sensory stimuli can modulate theorganization of the CPG to produce the appropriatebehavior.

In an effort to facilitate further comparative andevolutionary studies, similarities in structure, functionand pharmacology of certain potentially homologousneurons will be addressed in those species, in which thebuccal CPG organization has been analyzed most (He-lisoma, Planorbis, Lymnaea and Aplysia). Some of thedifficulties involved in interspecific comparisons will beaddressed and a plea made for more comparative studies.

2.1. Helisoma tri6ol6is as a neuroethological modelsystem

About 1970, an extensive survey of the gross anatomyof the buccal ganglia and associated structures involvedin feeding in various gastropods was made by S.B. Kater(personal communication). The purpose of this surveywas to find the preparation offering the best combinationof visual identifiability of buccal neurons and robustnessof feeding behavior in semi-intact preparations. Analbino strain of Helisoma tri6ol6is was selected anddeveloped as a neuroethological preparation for examin-ing feeding behavior. Hemoglobin inside glial cells pro-vided a red background contrasting with the white ‘polkadots’ of neuronal somata (cf. Pentreath et al., 1985).Simultaneous intracellular recordings from identifiableneurons and extracellular recordings from muscles couldbe made while the animal performed oral behaviors (e.g.Kater, 1974).

2.2. Feeding beha6ior and its anatomical basis inbasommatophoran snails

Helisoma and several other basommatophoran snailsare omnivorous with a variety of feeding mechanismsalthough, as a group, the basommatophora are morestereotyped in their feeding behaviors than are theopisthobranchs (cf. Audesirk and Audesirk, 1985;Thomas et al., 1985). The anatomy and/or dynamics ofthe feeding apparatuses have been studied in a numberof species (e.g. Hubendick, 1957; Demian, 1962; Hem-


brow, 1973; Dawkins, 1974; Kater, 1974; Goldschmed-ing and DeVlieger, 1975; Brace and Quicke, 1981;Smith, 1988, 1990; Arnett, 1996). All of these studiesdrew heavily on the work of Carriker (1946) onLymnaea stagnalis. In Helisoma feeding behaviors andtheir anatomical bases were initially examined by Kater(1974), who also tried to correlate neural motor pat-terns generated by the buccal ganglia with feedingbehavior. Smith (1988, 1990) extended Kater’s observa-tions on the anatomy and dynamics of the feedingapparatus. Later Arnett (1996) combined videomi-croscopy of buccal mass movements with simultaneousextracellular myograms and intracellular recordingsfrom motor neurons during oral behaviors in semi-in-tact preparations. She focused on the roles of threeprominent muscles of the feeding apparatus and theiractivity patterns, and identified motor neurons inner-vating these muscles. Specific neural patterns mediatingtypical feeding, regurgitation, and swallowing behaviorswere determined.

The mouth of Helisoma opens into a buccal cavitywithin a muscular pharyngeal buccal mass. The buccalmass consists of approximately 30 muscles, most occur-ring as bilateral pairs (Smith, 1990). The odontophore(tooth carrier) arises from the floor of the buccal cavity.The odontophore consists of a tongue-like ‘cartilage’and the attached file-like dentated radula. The chitinousradular teeth are secreted by the subradular membrane,which is continuous with the epithelial lining of the oralcavity. The odontophore cartilage is not true cartilagebut is a complex tissue including intrinsic muscle fibersand mechanical support structures.

These snails typically eat microflora by protractingthe odontophore forward and downward through themouth and rasping the radula over the substrate. Adja-cent to the mouth are one dorsal and two lateralchitonous mandibles. Helisoma cuts small pieces frommacrophytes by employing the dorsal lip of the odon-tophore and the dorsal mandible as ‘scissors’. Using theodontophore as a scoop with a moving radular con-veyor belt, these snails will also feed on loose material(e.g. in the surface film of ponds) and they also eatcarrion routinely. We have not observed Helisoma touse the buccal mass as a pump for ingesting semi-de-cayed matter without use of the odontophore, but thismethod was reported for the closely related snail Biom-phalaria (Thomas et al., 1985).

At rest, the odontophore sits at about a 45° angle.During a typical feeding cycle (Fig. 1), the tip (or dorsallip) of the odontophore is ‘protracted’ or rotated for-ward and downward and protruded through the mouth.During the second phase, the ondotophore is raspedover the substrate and ‘retracted’ back to the restposition within the oral cavity. The tip of the odon-tophore is kept in close apposition to the food groovealong the midline of the roof of the buccal mass during

retraction. In the third phase, the odontophore is hy-per-retracted, bringing the tip of the radula near theopening of the esophagus. If the snail is feeding slowly,the odontophore returns to the rest position during theinter-bite interval. During the protraction stroke, thefulcrum, about which the odontophore rotates, ismoved posteriorly such that the tip of the odontophoreis not in close apposition to the food groove. Onevariation of the typical feeding behavior is a sequenceof repetitive swallows, during which the dorsal lip ofthe odontophore is thrust or hyper-retracted repeatedlytoward the esophagus without intervening protractionsof the odontophore (Arnett, 1996).

3. Neurophysiological correlates of feeding in Helisoma

Neurophysiological studies have focused on the neu-ral basis of the rhythmic scraping or rasping move-ments of the odontophore described above. Thepioneering study of Kater (1974) influenced greatly notonly the subsequent studies of feeding in Helisoma, butalso comparative studies of feeding in other gastropods(e.g. Benjamin and Rose, 1979; Bulloch and Dorsett,1979). This influence had both positive and negative

Fig. 1. A schematic diagram of a feeding cycle in Helisoma. Theanterior of a snail is depicted from a lateral perspective as if therewere an optical sagital section through the mouth, odontophore(odontopore cartilage, OC, plus the attached radula, R), buccal mass(BM) and esophagus (E). In position 1, the odontophore rests atabout a 45° angle. The odontophore is protracted through position 2to the vertical position 3 with the lip of the odontophore protrudingthrough the mouth. The second odontophoral image in ‘3’ depicts theodontophore at the end of the rasp portion of the retraction phase.Position 4 depicts the odontophore near the end of the retractionphase. Position 5 shows the hyper-retracted odontophore, which hasbrought food particles to the opening of the esophagus.


Fig. 2. A schematic diagram of the original model of the Helisomabuccal pattern generator and the putative feeding motor pattern. Themotor pattern consisted of alternating bursts in retractor (R) motorneurons and ‘protractor’ (P) motor neurons (now known to behyper-retractor neurons). This pattern was thought to be generatedby a single neuronal oscillator comprised of approximately 20 pairs ofelectrotonically coupled interneurons, collectively called the ‘cyber-chron’ (C). The cyberchron was thought to drive and time the actionpotential bursts in motor neurons by exciting simultaneously theretractor neurons and inhibiting protractor neurons. The protractormotor neurons would then fire bursts of action potentials frompostinhibitory rebound. (After Kater, 1974).

myograms attributed to the posterior jugalis (pj), amajor odontophoral protractor muscle, were associatedon a one-to-one basis with action potentials in motorneuron B21. However, the pj muscle is very thin andoverlies the relatively massive supralateral radular ten-sor (slrt) muscle. It is difficult to distinguish pj activityfrom underlying tensor muscle activity when recordingfrom the intact buccal mass (cf. Rose and Benjamin,1979). Recently, feeding in Helisoma has been demon-strated to arise from a triphasic motor pattern (seebelow, Fig. 3; Arnett, 1996; Quinlan and Murphy,1996; Quinlan et al., 1995, 1997) corresponding to theprotraction, retraction, and hyper-retraction phases ofthe feeding cycle. The slrt muscle contracts duringretraction and even more vigorously during hyper-re-traction. The muscle potentials ascribed to the pj pro-tractor muscle and associated with neuron B21 actionpotentials by Kater (1974) were undoubtedly recordedfrom the underlying slrt. No morphological or otherdistinctive characteristics of neuron B21 were pub-lished, so it is not uniquely identifiable. However thelarge neurons B18 and B19 displayed similar patterns ofsynaptic and action potentials to the neuron designatedB21 by Kater (1974). Neurons B18 and B19 innervatethe slrt, and during feeding motor activity, they fire

aspects. A series of ‘mistakes’ by Kater (1974) illus-trates generally applicable pitfalls in correlating neu-ronal activity with corresponding evoked muscleactivity in relatively complex neuromuscular systems. Avisual interpretation of the feeding behavior, combinedwith the misidentification of the muscular target of aneuron, led to a miscorrelation of activity in a set ofneurons with the protraction phase of the feeding cycleand further led to the conclusion that the feeding cycleand its underlying neural pattern were biphasic ratherthan triphasic. Therefore, I will explain the reason forand the consequences of, the mistakes made in consid-erable detail.

During typical rapid rhythmic feeding in Helisoma,there is no pause at the ‘rest’ position between theretraction and hyper-retraction phases, and protractionfor the subsequent bite cycle commences immediatelyupon termination of the hyper-retraction phase. There-fore, Kater (1974) separated the feeding cycle into onlytwo phases — protraction, and retraction. Katerrecorded a biphasic buccal motor pattern with alternat-ing bursts of action potentials in two sets of motorneurons (e.g. Fig. 2). The bursts of action potentials inone set of motor neurons (represented by neuron B27,see Fig. 3) were correctly correlated visually with theretraction phases of the feeding cycles, and hence, thisset of motor neurons was designated ‘retractor motorneurons’ (Kater, 1974). Since there was thought to beonly a biphasic pattern, the remaining phase of neuralactivity recorded was taken to represent protraction,and neurons generating action potentials during thispart of the cycle were designated ‘protractor motorneurons’ (e.g. neurons B17, B18, B19, B20, and B21;Kater, 1974).

The correlation of the neural pattern and feedingbehavior appeared to be confirmed when extracellular

Fig. 3. The triphasic feeding pattern of buccal motor neuron activityin Helisoma. Simultaneous intracellular recordings from identifiedbuccal motor neurons demonstrated three discrete phases (1, 2, 3) ofexcitation in each of the four cycles of feeding activity depicted.Protractor motor neuron B6 was depolarized during phase 1, andinhibited during phase 2. Retractor motor neuron B27 was depolar-ized during phase 2, and Hyper-retractor motor neuron B19 wasinhibited during phase 2 and depolarized during phase 3. Note, thatthe pattern depicted in Fig. 2 corresponds to phases 2 and 3 of thefeeding pattern.


bursts of action potentials during the hyper-retractionphase (Arnett, 1996). Careful dissection of the buccalmass, allowing myograms to be recorded from the pjprotractor muscle independently of the slrt, revealedactivity in pj myograms occurring after the hyper-re-tractor activity (e.g. B19 bursts) of the previous feedingcycle and prior to retractor activity in, for instanceneuron B27. Subsequently, it was demonstrated thatprotraction phase neurons B6 and B8 innervate the pj(see below; Arnett, 1996). Thus the bi-phasic electro-physiological pattern described by Kater represents thesecond and third phases (i.e. retraction and hyper-re-traction) of the triphasic feeding pattern (compare Figs.2 and 3). The activity that Kater associated with pro-traction actually mediates hyper-retraction of the odon-tophore to facilitate swallowing.

3.1. The original model of the Helisoma buccal CPG

The retractor motor neurons described by Kater(1974) displayed excitatory postsynaptic potentials (EP-SPs) underlying their action potential bursts. Simulta-neously, the ‘protractor’ (actually hyper-retractor andradular tensor) motor neurons displayed prominentinhibitory postsynaptic potentials (IPSPs). Electrotoniccoupling among synergistic motor neurons was demon-strated. A model of the buccal CPG (Fig. 2) wasdeveloped whereby only a single neural oscillator wouldsimultaneously evoke EPSPs in the retractor motorneurons and IPSPs in the ‘protractor’ motor neurons.The protractor motor neurons would subsequently gen-erate a burst of action potentials from post-inhibitoryrebound. Indeed, the ability of these motor neurons togenerate a vigorous burst of action potentials followingtermination of a hyperpolarizing pulse wasdemonstrated.

A set of an estimated 20 or so pairs of electrotoni-cally coupled neurons was thought to drive and timethe activity of the motor neurons, and thus, were called‘cyberchron neurons’. These neurons seemed to meetthe criteria for comprising the feeding CPG. Theseneurons evoked PSPs of the appropriate sign for theretraction phase of the feeding cycle in a number ofidentified motor neurons. A brief stimulation of a cy-berchron neuron could sometimes trigger a number ofcycles of rhythmic activity in feeding motor neurons.However, action potential bursts typically were notrecorded from cyberchron neurons during sustainedrhythmic activity (e.g. Kater, 1974). Cyberchron neu-rons did, however, display typically the depolarizationscoinciding with the synaptic activity in motor neuronsduring the retraction phase of the cycle. Therefore, itwas hypothesized that a few neurons of the electrotoni-cally-coupled network of approximately 20 pairs ofcyberchron neurons were generating action potentials

during any given feeding cycle and, by chance, theactive neurons were not recorded.

Electrotonic coupling and its plasticity were studiedextensively in this network (Kaneko et al., 1978; Mer-ickel and Gray, 1980). The potential roles of electro-tonic coupling during rhythmic action potential burstgeneration in a reverberating coupled system were ex-amined by computer modeling (Merickel et al., 1977,1978). Some members of the cyberchron network werealso shown to have ‘bursty’ membrane properties (i.e. aregion of negative slope resistance in their I–V curve).Later, however, it was shown that the IPSPs evoked in‘protractor’ (i.e. hyper-retractor) motor neurons by ac-tion potentials in cyberchron neurons reversed at mem-brane potentials much more depolarized than thereversal potential of the IPSPs evoked by the buccalCPG during phase 2 (i.e. retraction) of the feeding cycle(Murphy, 1991). More recently, it has been shown thatactivity evoked in some ‘cyberchron neurons’ triggersregurgitation. Thus, this group of neurons has beenredesignated the Buccal A Cluster (BAC) neurons (Ar-nett, 1996) since they are not directly the componentsof the buccal CPG and they neither time nor drive theactivity of motor neurons during feeding.

3.2. A triphasic buccal motor pattern underlies feedingbeha6ior

A triphasic buccal motor pattern (Fig. 3) was de-scribed in Helisoma (Quinlan and Murphy, 1991, 1996;Quinlan et al., 1995, 1997). It is quite similar to pat-terns reported to underly feeding in the basomma-tophoran snail, Lymnaea (Elliott and Benjamin, 1985a)and the opisthobranch, Tritonia (Bulloch and Dorsett,1979). Since the Helisoma buccal ganglia can produce anumber of different motor patterns, this triphasic pat-tern was designated the ‘standard pattern’ of buccalmotor activity and was hypothesized first (Quinlan andMurphy, 1991) and later, demonstrated to underly thetypical feeding (Arnett, 1996). This pattern allowedneurons to be classified as active in one or more specificphase(s) of the feeding pattern. A number of neuronsactive in each of the three phases has been identified(see Figs. 4 and 14 and Table 1).

Protraction phase motor neurons (or presumptivemotor neurons) include neurons B3, B6, B7, and B8(Fig. 5). These neurons are excited during phase 1 andinhibited during phase 2. They recover slowly from thephase 2 hyperpolarization, remaining inhibited throughphase 3. The physiology of neuron B3 differs from thatof neurons B6, B7, and B8. In rapid feeding rhythms, itfires bursts of action potentials only during phase 1.However, if there is a slow feeding rhythm, neuron B3fires a burst of action potentials not only during phase1, but also in the interphase following phase 3. If thereis rhythmic retraction and hyper-retraction phase activ-


Fig. 4. A map of the somata of identified Helisoma buccal neurons.Both the caudal surface and the rostral surface of the animal’s leftbuccal ganglion are depicted. Each identified neuron would have amirror image homolog in the right ganglion (not shown). Note, thatsome terminology is altered from previous publications. The rostraland caudal surfaces were previously called ventral and dorsal, respec-tively; the VBN and LBN were previously designated the HBN andVBN, respectively (Kater, 1974). Neurons on the rostral surface werenamed formerly VB1-VB10 (to indicate ventral buccal neurons;Lukowiak and Murphy, 1987) and they have been renamed 101–110.The changes were made to conform to the in situ morphology of thestructures, and to prior terminology of basommatophoran molluscanmorphologists (e.g. Carriker, 1946; Hembrow, 1973), and to facilitatecomparisons with other gastropods. BC, buccal commisure; CBC,cerebrobuccal connective; ET, esophageal nerve trunk; LBN, later-obuccal nerve; PBN, posterobuccal nerve; VBN, ventrobuccal nerve.

Fig. 5. Morphology of identified Helisoma phase 1 protractor motorneurons. Photographs of neurons injected with the fluorescent dye,Lucifer Yellow. All of the neuronal somata are on the caudal surfacesof the buccal ganglia. Orientation of ganglia is the same as in Fig. 4.Influential motor neuron B6 (top) has axons in both PBNs andinnervates the pj muscle bilaterally. Neuron B7 (middle) has anextensive neuritic arbor in the ipsilateral buccal ganglion and a singleaxon that traverses the ipsilateral VBN. Neuron B3 (lower left) wasphotographed in the right buccal ganglion. It sends a proximal axonsegment toward the CBC. The axon makes a sharp turn to traversethe esophageal trunk to the dorsobuccal nerve branch (not shown),which it then follows into the buccal mass. Neuron B8 was pho-tographed in the left buccal ganglion and has an axon only in theipsilateral PBN. It innervates the ipsilateral pj. Calibration bar, 100mM.

ity but no protraction phase activity, neuron B3 fires aburst of action potentials following the hyper-retractionphase. The specific muscular target(s) of neuron B3have not been identified, but they may help return theodontophore to the rest position. Neuron B3 projectsan axon to the buccal mass via the ipsilateralesophageal nerve trunk (ET) and one of its branches,the dorsobuccal nerve (DBN). Neurons B6 and B8innervate the pj muscle bilaterally and ipsilaterally,

Table 1The sign of PSPs evoked in identified Helisoma effector neurons byeach CPG subunita

Axonal projectionsNeuron S2S1 S3

Ip and con Ets (GNs, DBNs)IB5 I IIp and con Ets (SNs, DBNs)EB4 E

E EB34 Ip LBNE IB3 Ip ET (DBN)

B6 Ip and con. PBNsIEI Ip VBNEB7

B8 E I Ip PBNIp LBNB17 I E

IB19 E Ip and con VBNs, ip LBNB18 I/E E Ip and con VBNs

EB26 E Ip and con VBNsEB27 E Ip and con LBNs

B29 E Ip and con PBNsIp and con VBNs and LBNsEB110

a I, inhibitory; E, excitatory; Ip, ipsilateral; con, contralateral; ET,esophageal trunk; GN, gastric nerve; DBN, dorsobuccal nerve; SN,salivary nerve; VBN, ventrobuccal nerve; PBN, posterobuccal nerve;LBN laterobuccal nerve.

respectively, via the posterobuccal nerves (PBNs). Ax-ons of neuron B7 enter the buccal mass via the ipsilat-eral ventrobuccal nerve (VBN; previously known as theheterobuccal nerve, see the legend of Fig. 4 for explana-tion of nomenclature). Neuron B34 is excited in phases


1 and 2 and can have its highest spike frequency ineither phase, depending on conditions. Neuron B34 hasan axon in the ipsilateral LBN.

Retraction (phase 2) motor neurons and presumptivemotor neurons include neurons B110 (aka VB10), B26,B27, and B29 (Fig. 6). Neurons B29 and B110 generatebursts of action potentials only during phase 2. NeuronB29 has axon branches in both PBNs that pass throughthe pj muscle (but do not innervate it) and continue tounidentified targets deeper in the buccal mass. NeuronB110 has bilateral axon branches in both VBNs andLBNs. It innervates the slrt muscle (M. Zoran, personalcommunication). Neurons B26 and B27 are excitedstrongly by S2 interneurons and more weakly by S3interneurons. During slow motor patterns, neuron B27

Fig. 7. Current model of the Helisoma buccal CPG. Squares representthe three interneuronal subunits (S1, S2, and S3) of the CPG. Eachsubunit is a conditional oscillator that serves as a unit patterngenerator and provides the primary excitation to a correspondingsubset of buccal motor neurons (S1MN, S2MN, and S3MN). Linesending in perpendicular bars indicate excitatory connections, andthose ending in closed circles indicate inhibitory connections. Arrowsindicate that both the oscillatory properties of each subunit, and thesynaptic connections between subunits, are potential targets of modu-lation.

Fig. 6. Morphology of phase 2 retractor motor neurons. NeuronB101 (top) was viewed and photographed on the rostral surfaces ofthe buccal ganglia. It has an extensive neuritic arbor in both gangliaand in the buccal commissure. It is bipolar and has axons in ipsi- andcontralateral VBNs and LBNs. Neurons B27 (middle) and B29(bottom) were viewed and photographed on the caudal surfaces ofthe buccal ganglia, with orientation as in Fig. 4. Neuron B27 has anextensive ipsilateral neuritic arbor and has axons that traverse theipsi- and contralateral LBNs. Influential motor neuron B29 (bottom)has axons in both PBNs that pass through the pj muscle to unknowntargets. Calibration bar, 100 mM.

displays a rapid burst of action potentials in phase 2and fires more slowly in phase 3 (Quinlan and Murphy,1996). During a vigorous feeding pattern, neuron B27generates bursts of action potentials confined almostentirely to phase 2 of the pattern because it displays apostburst hyperpolarization that inhibits its firing dur-ing phase 3 (e.g. Fig. 3). Neuron B26 has axons inipsilateral and contralateral VBNs and innervates theslrt muscle and Neuron B27 has axons in the ipsilateraland contralateral LBNs and innervates the aj muscle.

Hyper-retraction (phase 3) motor neurons includeneurons B17, B18, and B19. Neuron B17 has an axonin the ipsilateral LBN but specific muscular targetshave not been identified. Neurons B18 and B19 haveaxons projecting bilaterally to innervate the slrt musclevia the VBNs (aka HBNs). Neuron B19 also innervatesthe lateral portions of the ipsilateral aj muscle via theipsilateral LBN, and in some preparations, there is anaxon in the contralateral LBN and a bilateral innerva-tion of the aj (Arnett, 1996).

3.3. The current model of the multifunctional buccalCPG

The CPG consists of three semi-independent in-terneuronal subunits, S1, S2, and S3, each of which isan oscillator capable of generating rhythmic bursts ofaction potentials or, in the case of S2, rhythmic plateaupotentials (Fig. 7 and see Quinlan et al., 1995). Eachsubunit provides excitation or inhibition to subsets ofbuccal motor neurons. Some motor neurons are excited(or inhibited) by more than one subunit (Table 1) and,thus, can display action potential bursts, which span all


or part of the duration of more than one phase of thecycle (Fig. 3 and cf. Quinlan and Murphy, 1996).Therefore, (and for additional reasons) a number ofmotor neurons have action potential bursts that overlapin time and do not fit discretely into only one of thethree phases of the feeding cycle. In the feeding mode,S1 interneurons stimulate protraction (phase 1) motorneurons and S2 interneurons. This excitation brings S2interneurons to threshold for plateau potential genera-tion. S2 interneurons provide feedback inhibition ontoS1 interneurons, terminating their activity. Simulta-neously, S2 plateau potentials inhibit S3 interneurons.When the inhibition subsides, S3 interneurons fire frompost-inhibitory rebound. Thus, feeding results from anS1-S2-S3 sequence of activity. As long as there is tonicexcitation to S1 interneurons or an excitatory milieu ofneuromodulators (e.g. high dopamine concentrations),rhythmic feeding activity will continue (Quinlan et al.,1997).

3.4. Plasticity of organization in a multifunctional CPG

A flexible organization of the multifunctional buccalCPG was suggested by the analyses of many simulta-neous intracellular recordings of two to four neuronsfrom a set of 20 or so frequently monitored identifiedneurons. Sets of PSPs, characteristic in sign and ampli-tude of PSPs occurring during a given phase of thetriphasic feeding motor pattern, were recorded frommultiple neurons and often occurred either sporadi-cally, or rhythmically, but in the absence of a fulltriphasic feeding pattern. These phase-characteristicPSPs were correlated consistently across buccal neu-rons, suggesting that CPG interneurons could be acti-vated in functional motor patterns other than thetriphasic pattern, or perhaps in non-functional degener-ate motor patterns.

However, the existence of correlated PSPs, that ap-pear to be the characteristic of those of a particularphase of the standard feeding pattern, does not indicatenecessarily that these PSPs arise from feeding CPGinterneurons. For example, the BAC neurons (aka cy-berchron neurons, Kater, 1974) are not a part of thebuccal CPG but can evoke widespread PSPs of the signappropriate for phase 2 PSPs. Therefore, to identifyinterneurons definitively as components of a multifunc-tional buccal CPG and to show that modulation oftheir activities could result in multiple motor patterns,it was necessary to show, (1) that they were activeduring a particular phase of the triphasic feeding pat-tern; (2) that stimulation of putative interneurons inquiescent preparations evoked phase-characteristicpostsynaptic potentials (PSPs) in a set of follower neu-rons; (3) that hyperpolarization of the putative in-terneurons during a feeding pattern eliminatedappropriate phase-specific PSPs; (4) that brief hyperpo-

larizations of interneurons could reset the phase ofmotor patterns; and (5) that activity of the putativeinterneurons during non-feeding motor patterns elicitsPSPs similar to the phase-characteristic PSPs evoked bythe interneurons during the feeding motor pattern. Anumber of CPG interneurons have been identified andtheir activity patterns confirmed the flexible organiza-tion of the multifunctional pattern generator (cf. Quin-lan and Murphy, 1996).

3.5. Identified CPG interneurons and motor neurons

Commonly for invertebrates, it is impossible to pi-geon-hole neurons as definitively ‘motor neurons’, ‘sen-sory neurons’ or ‘interneurons’ since a given neuronmay function in multiple roles (e.g. Miller and Selver-ston, 1985; Evans et al., 1996; Hurwitz et al., 1996).With that caveat, in Helisoma, identified neurons activein each of the three phases of feeding could be catego-rized as pure interneurons, influential neurons, or fol-lower neurons on the basis of function andmorphology. Central pattern generator (CPG) elementsinclude pure interneurons and influential neurons (cf.Arshavsky et al., 1988a). Pure interneurons either pro-ject axons to the cerebral ganglia (BCN1s) or have theirprocesses confined to the buccal ganglia and the proxi-mal areas of buccal nerve roots (e.g. N1a, N1b, N1c).They do not project axons to peripheral targets. Influ-ential neurons are multifunctional neurons that haveperipheral axons with either sensory or motor functionsbut which also participate in the generation and/ormodulation of buccal motor patterns. Influential neu-rons can play roles in the CPG that are as critical andprominent as those of pure interneurons.

Phase 1 pure interneurons include three pairs ofBCN1 neurons, with axons crossing the buccal commis-sure and traversing the contralateral cerebrobuccal con-nective with axonal arbors both in the buccal andcerebral ganglia. In addition, neurons N1a, N1b, andN1c have processes confined to the buccal ganglia orproximal areas of buccal nerves (Fig. 8). Neuron B6 isa phase-1 influential neuron that also serves as a motorneuron innervating the pj muscle (Arnett, 1996). Stimu-lation or hyperpolarization, respectively, of neuron B6can evoke or inhibit the feeding motor pattern.

Central pattern generator (CPG) interneurons evok-ing retraction phase (i.e. phase 2) PSPs during feedingare the pair of glutamatergic interneurons, B2 (Quinlanet al., 1995). Interneuron B2 is a phase 2 influentialneuron, activity in which appears necessary and suffi-cient to account for S2-evoked PSPs. Neuron B2 has anaxon, which traverses the buccal commissure and formsa loop and returns to the ipsilateral neuropile. Neuriticprocesses arborize in both buccal ganglia and axonbranches also project to the buccal mass via the poste-rior buccal nerves. Functions of the peripheral axons of


neuron B2 are unclear and could include sensory ormotor roles (Quinlan et al., 1995). Neuron B29 is aphase-2 influential neuron, stimulation of which canevoke phase 2-like PSPs in other buccal motor neurons(cf. Figs. 3, 6 and 9). However, hyperpolarization ofneuron B29 does not eliminate phase-2 activity in otherneurons. No ‘pure interneurons’ with axons confined tothe CNS and active in phase 2 have been described inHelisoma. However, Arshavsky and colleagues reported‘group 2’ interneurons in the snail, Planorbis, (likeHelisoma in the family Planorbidae) that were active inphase 2 of the feeding cycle and had no processesprojecting from the buccal ganglia (Arshavsky et al.,1988a).

The major elements of subunit 3 of the Helisomabuccal CPG are the pair of pure buccal interneuronsN3a (Quinlan and Murphy, 1996). Activity in neuronsN3a is necessary and sufficient for phase-3 PSPs duringthe triphasic feeding pattern and also accounts forphase 3-like PSPs occurring in non-feeding patterns. Acluster of S3 influential neurons (B101–104) that areimmunoreactive to small cardioactive peptide B (Fig.10) have somata on the rostral (aka ventral) surfaces ofthe buccal ganglia and axons in the posterior buccalnerves (Lukowiak and Murphy, 1987). In addition totheir roles as S3 CPG elements, these neurons functionas radular mechanoafferents (unpublished data). Theycompensate apparently for load on the radula by acti-vating S3 interneurons, and thus, the hyperretractionmotor neurons.

3.6. E6idence that the triphasic standard buccal motorpattern mediates the typical feeding beha6ior

Since several different rhythmic motor patterns wererecorded from identified buccal neurons in Helisoma, itwas necessary to determine which neural pattern medi-ated the typical rasping behavior described above. Thisrequired semi-intact preparations, in which an incisionwas made along the dorsal midline of the head region,the body wall was reflected outward, and the esophagusand extrinsic buccal retractor muscles were severed toallow the buccal mass to be tilted forward. Thisbrought the buccal ganglia, situated on the caudalsurface of the buccal mass, into a dorsal position. Amicroplatform placed underneath the buccal gangliastabilized the preparation for intracellular recordingswhile the buccal mass was free to perform feedingmovements (Arnett, 1996; Arnett and Murphy, 1991,1992). A reasonably good facsimile of feeding behaviorcould be evoked by feeding stimulants, with the caveatthat incomplete protraction of the odontophore wasseen, possibly due to a loss of hydrostatic pressurewithin the body cavity and within the buccal mass. Thetriphasic motor pattern (Fig. 3) was simultaneouslyrecorded intracellularly from identified neurons andextracellularly from specific muscles of semi-intact adultsnails performing feeding-like movements of the odon-tophore. However, the position of the odontophorecould only be seen during part of the feeding cycle insemi-intact adults. It was, therefore, necessary to defineexternal features of the buccal mass that were thediagnostic for the position of the odontophore duringeach phase of the feeding cycle. Newly hatched snails(about 1 mm in shell diameter) were videotaped whilefeeding on glass slides or transparent plastic sheets.Such small snails are semi-transparent and the positionsof the odontophore were correlated readily with exter-nal features of the buccal mass. For any given phase ofthe feeding cycle, the positions and overall contours of

Fig. 8. Morphology of protraction phase S1 interneurons. Interneu-rons were stained by intracellular injection with Lucifer Yellow.Tracings were made from sets of Ektachrome slides of each neurontaken at multiple focal planes and superimposed. There are six pairsof identified S1 interneurons. The BCN1 group consists of three pairsof similar neurons that are not readily distinguishable from eachother. These neurons send axons across the buccal commissure andtraverse the contralateral CBC (upper left) to form a terminal arborin the cerebral ganglion. Often, a short neuritic process extends intoone or more of the esophageal trunks (as shown here) or posterobuc-cal nerves. Neurons N1a, N1b and N1c do not have axons leaving thebuccal ganglia though N1b has processes extending a few hundredmicrometers into the CBCs.


Fig. 9. Neuron B29 is a retraction phase influential neuron. Simultaneous intracellular recordings were made from neuron B19, which is inhibitedduring phase 2 and excited during phase 3 of the feeding pattern. Activity in neuron B29 was recorded with a high resistance unbalanced LuciferYellow electrode. At the beginning of the record neuron B29 was held hyperpolarized (0.8 nA) and neuron B19 was firing tonically. Neuron B29was released periodically from hyperpolarization, which evoked anode break bursts of action potentials in neuron B29 that triggered phase 2-likeIPSPs in neuron B19 (labeled ‘2’ in the second series of anode break bursts). Spontaneous bursts in B29 coincided with IPSPs in B19, and adepolarizing pulse ‘D’ injected into neuron B29 evoked a phase 2-like IPSP in neuron B19. Calibrations, 10 s, 40 mV.

the buccal mass, as well as the degrees of protrusion ofthe radular sac were distinctive. Since similar positionsand contours of the buccal mass and radular sac wereseen during feeding behaviors of semi-intact adults, itwas possible to correlate the stages of the feeding cyclewith the appropriate phases of the triphasic neuralpattern. The aj, pj, and slrt muscles are prominentmuscles of the buccal mass that are visible when themass is viewed from an external perspective. Theirstates of contraction or relaxation are major determi-nants of the positions of the odontophore and of thecontours of the buccal mass during the three phases ofthe feeding cycle (Arnett, 1996).

The pj muscle contracts during protraction of theodontophore and different regions of the aj and slrtmuscles contract both during retraction and hyper-re-traction. Neurons B6 and B8 innervate the pj, bilater-ally and ipsilaterally, respectively, and generate burstsof action potentials during phase 1 of the feedingpattern. Neuron B27 generates action potential burstsprimarily during phase 2 and innervates the medialportion of the aj muscle, which contracts during retrac-tion. Neuron B19 displays action potential bursts dur-ing phase 3 of the standard buccal motor pattern andinnervates the lateral regions of both the aj and slrtmuscles, which contract vigorously during hyper-retrac-tion of the odontophore. Thus, identification of motorneurons for the aj, pj, and slrt muscles confirmed boththe roles of these muscles during feeding and that thestandard triphasic buccal motor pattern mediates themost typical feeding behavior (Arnett, 1996).

3.7. Regurgitation

Regurgitation can be triggered in basommatophoransnails by feeding them noxious materials (Bovbjerg,1968). We observed serendipitously that Listerine is aneffective emetic. This observation was exploited to trig-ger regurgitation in intact newly hatched snails, inwhich the dynamics of the buccal mass and movementsof the odontophore could be observed (Arnett, 1996;Arnett and Murphy, 1994).

Microscope slides were coated with agar containingwatermelon extract, a potent feeding stimulant. Lister-ine was applied via Pasteur pipettes near the mouths ofnewly hatched snails feeding vigorously on the water-melon-coated slides. These snails would perform imme-diately one or several odontophore reversals (i.e.regurgitation strokes). A number of features distin-guished regurgitation cycles from feeding cycles. The

Fig. 10. Morphology of phase 3 interneuron N3a and influentialneuron B101. Tracings of Lucifer Yellow stained neurons made fromEktachrome slides taken at multiple focal planes. Neuron N3a (top)is located on the caudal surface of the buccal ganglia. Neuron (B101)lower is the largest of a cluster of small cardioactive B immunoreac-tive neurons on the rostral surface of the buccal ganglia. It has shortneuritic processes in the proximal ipsilateral CBC and ET. It hasaxons in both PBNs and functions as a radular mechanoreceptor.


power stroke of the odontophore switched from retrac-tion during feeding to protraction during regurgitation.During feeding, the fulcrum of the odontophore (i.e.the point about which the odontophore rotates) ismoved forward at the beginning of the retractionstroke. Thus, the dorsal lip of the odontophore isapposed closely to the lining of the oral groove duringretraction and food particles are swept posteriorly.During regurgitation, the fulcrum of the odontophoremoved forward at the beginning of protraction so thatthe lip of the odontophore was apposed to the oralgroove during protraction, thus sweeping food particlesout the mouth. The fulcrum, then, moved backward atthe beginning of retraction so that the lip of the odon-tophore was not apposed to the oral groove duringretraction. Additionally, the protraction phase (i.e.phase 1) was prolonged during regurgitation. Finally,there was, typically, no hyper-retraction of the odon-tophore during regurgitation cycles. Only the protrac-tion/retraction phases of the trajectory were displayed.

The physiological correlates of regurgitation includedprolonged phase 1 bursts of action potentials (underly-ing the protraction phase), relative to those seen duringfeeding, and a suppression of S3 activity, therefore,eliminating hyper-retraction. The neural pattern under-lying regurgitation was examined in semi-intact adultsnails, in which the esophagus was canulated withpolyethylene tubing connected via a two-way valve toseparate syringes containing watermelon extract or 25%listerine (Arnett, 1996; Arnett and Murphy, 1994).Feeding behavior and the triphasic feeding motor pat-tern could be triggered in quiescent snails by perfusingthe oral cavity with watermelon extract. Switching thevalve to admit 25% listerine into the oral cavity, trig-gered a number of strong regurgitation cycles, withvigorous protraction of the odontophore, interspersedwith occasional weak regurgitation cycles with onlypartial protraction of the odontophore. The strongprotraction cycles were correlated with the prolongedaction potential bursts in S1 interneuron N1a, relativeto those seen during feeding. Neither hyper-retractionof the odontophore, nor phase 3 action potential burstsin motor neuron B19 were observed.

3.8. Transmitters of CPG interneurons

Here, we will focus on the neurotransmitters, do-pamine, GABA and glutamate because their roleswithin the Helisoma buccal CPG are best characterized.Exogenous application of 5–10 mM dopamine elicitsthe triphasic feeding motor pattern in Helisoma reli-ably. An endogenous source of dopamine is phase 1interneuron N1a of the buccal CPG (Quinlan et al.,1997). Stimulation of interneuron N1a by current injec-tion evokes the triphasic feeding pattern. N1a is anintrinsic element of the CPG. A brief hyperpolarization

Fig. 11. GABA excites protraction phase motor neurons and evokesrhythmic activity in CPG subunit 2 by pharmacologically distinctmechanisms. GABA triggered rhythmic action potential bursts inter-rupted by phase 2 IPSPs in the influential protractor motor neuronB6. Neuron B6 fired tonically in normal physiological saline (toptrace). At the first arrowhead, 100 mM was superfused over thepreparation. At the second arrowhead, the GABA was washed outwith normal saline. The GABAB receptor agonist, baclofen, triggeredrhythmic S2 activity as indicated by the phase 2 IPSPs in neuron B6,but it had no depolarizing effect on neuron B6. Muscimol, a GABAA/

C receptor agonist depolarized phase 1 motor neuron B6 but did nottrigger rhythmic activity in subunit 2. Calibrations 40 mV, 10 s.

of neuron N1a during a feeding pattern can reset thephase. The dopamine antagonist, sulpiride, blocks theeffects of both dopamine application and of neuronN1a stimulation. Interneuron N1a also stains with theformaldehyde/gluteraldehyde histochemical stain that isindicative of catecholamines and is highly specific fordopamine in molluscan nerve cells. Application of feed-ing stimulants (e.g. watermelon extract) triggers activityin neuron N1a and consequent feeding behavior.

GABAergic interneurons are also elements of S1 ofthe buccal CPG. Two of the three pairs of BCN1neurons are GABA immunoreactive and application ofGABA has a number of effects on identified buccalneurons (Richmond et al., 1991, 1994; Murphy, 1993).GABA depolarizes a number of identified protractionphase neurons that are excited by S1 interneurons (Fig.11). This effect is mimicked by muscimol and blockedby picrotoxin as one would expect if GABAA orGABAC-like receptors were activated. This excitatoryeffect of GABA is enhanced by chloride injection intothe phase-1 motor neurons suggesting that they nor-mally have a high concentration of chloride ionsinternally.

Either stimulation of BCN1 interneurons or superfu-sion of GABA also triggers rhythmic S2 activity. Thisactivation of rhythmic S2 activity is mimicked by ba-clofen and is not affected by picrotoxin, suggesting thatit is mediated by a GABAB-like mechanism. Finally,bath application of GABA (500 mM–1 mM) hyperpo-larizes phase 3 interneuron N3a as well as a number ofphase 3 motor neurons (Richmond et al., 1994). This


inhibitory effect is also mimicked by muscimol andblocked by picrotoxin. Thus, at these concentrations,GABA evokes an S1–S2 pattern of activity similar tothe pattern seen during regurgitation.

The primary phase 2 PSPs arise from glutamatergicinterneuron B2 (Quinlan et al., 1995). Interneuron B2has been co-stained by injection of the dye LuciferYellow and by immunocytochemical staining with glu-tamate antibodies. The phase 2 EPSPs evoked by stim-ulation of neuron B2 are mimicked by Kainate/AMPAand are blocked by CNQX (Quinlan and Murphy,1991). The phase 2 IPSPs are mimicked by quisqualateand have no known antagonist. Since glutamate isconsidered generally an excitatory transmitter, neuronB19 was placed in isolation in acute cell culture todetermine if the inhibitory effect of glutamate wasdirect or indirect. Glutamate or quisqualate applied tothe isolated neurons B19 directly evoked hyperpolariz-ing responses.

3.9. Sensory and modulatory pathways regulating thebuccal CPG

Sensory or modulatory afferents important for oralbehaviors impinge upon the buccal ganglia from one oftwo general pathways. Information from thesupraesophageal ring of ganglia (i.e. the ‘brain’) reachesthe buccal ganglia via the cerebrobuccal connectives(CBCs). Information from the buccal mass, oral cavity,odontophore, salivary glands and esophagus can reachthe buccal ganglia via buccal nerve roots that innervatethe buccal mass, salivary glands and esophagus.Mechano- and chemosensory afferents from the lips,oral veil and tentacles affect mainly the buccal gangliavia axons to the cerebral ganglia in the lip and tentacu-lar nerves.

Patterned motor activity evoked by chemosensorystimulation of the Helisoma oral cavity with feedingstimulants (10% wheat germ or 20% fish meal) wasdemonstrated by Horwitz and Senseman (1981). Can-nulae were placed in the mouth and in the esophagusand substances were perfused from the mouth throughthe oral cavity and esophagus. Examination of thefigures shows that rhythmic activity occurred in S2 andS3, indicated by IPSPs in neuron B19, similar to S2-evoked IPSPs, and action potential bursts in neuronB19, similar to those evoked by S3. However, theactivity in S2 and S3 was not phase-locked into theS2–S3 sequence, as it is during the typical feedingpattern and S1 activity was not monitored. Sensemannoted (personal communication) that there were rhyth-mic contractions of the buccal mass but it was difficultto tell if they were true feeding movements and theexperimental protocol forced the feeding stimulantsthrough the oral cavity from mouth to esophagus.

Perfusion of the oral cavity with a feeding stimu-lant (e.g. watermelon extract) via canulation of theproesophagus evoked the triphasic feeding patternand feeding movements (Arnett, 1996; Quinlan et al.,1997; see above). Feeding was evoked even if therewere no neural connections between the buccal gan-glia and the rest of the central nervous system.Hence, afferent information must have reached thebuccal ganglia via the buccal nerve roots. A numberof possibilities could account for the differences in thenature of the motor patterns observed by Horwitzand Senseman (1981), Quinlan et al. (1997). Perhapsa cannula in the mouth triggers mechanoafferentsthat interfere with the generation of the triphasicfeeding pattern. Alternatively, differences in thechemostimuli may have contributed to the differencesin observed motor patterns.

3.10. Higher order modulation of the CPG

Two descending modulatory elements have beencharacterized in Helisoma — the pair of giant sero-tonergic interneurons C1 (Granzow and Kater, 1977;Granzow and Rowell, 1981; Murphy et al., 1985a), andthe inhibitory pleurobuccal interneurons Pl1 (Murphy,1990). Neuron C1 is excited by feeding stimulants ap-plied to the lips and oral veil. Stimulation of neuronC1, or bath application of 1 mM serotonin, evokesconsistently the rhythmic phase-locked activity in S2and S3 of the buccal CPG. Serotonin can, but usuallydoes not, activate the full triphasic feeding pattern. Theeffects of serotonin on S1 activity are variable and anumber of qualitatively different motor patterns (alldisplaying linked S2–S3 activity) can be evoked byserotonin application (Quinlan and Murphy, 1996). AnS2–S3 pattern without activity in S1 mediates rhythmicswallowing (Arnett, 1996).

Patterned activity of the buccal CPG is inhibitedrapidly and completely either by exogenous applicationof the neuropeptide FMRFamide (Murphy et al.,1985b), or by stimulation of the putatively FMR-Famidergic interneuron Pl1 (Murphy, 1990). The so-mata of paired interneurons Pl1 are on the ventralsurfaces of the pleural ganglia. The main axon ofneuron Pl1 traverses the pleuropedal commissure, thepedocerebral commisure, and the cerebrobuccal com-missure to reach the buccal ganglia. It has neuriticarbors in the parietal, pleural, pedal, cerebral and buc-cal ganglia. Thus, it is perfectly situated to integrate avariety of stimuli and coordinate activity in the feedingand locomotory CPGs and to activate the whole bodywithdrawal response. Whether it performs all of theseintegrative roles remains to be determined (but seebelow).


4. Comparisons of gastropod neurons and feeding cir-cuitry

Two key related questions need to be addressed.1. Is the model of the buccal CPG, developed to

account for data from Helisoma and discussedabove (or, for that matter, any other model), gener-alizable to provide a universal model for gastropodbuccal neuronal organization?

2. Are buccal neurons and their interconnections con-served sufficiently to allow homologies of identifiedneurons to be ascertained and the evolution ofcircuitry at the cellular and subcellular levels to beanalyzed?

The tentative answer to both questions is yes, forthose gastropods with rasping or grasping radular feed-ing modes. However, more comparative data areneeded for a definitive answer.

The key feature of the Helisoma CPG organization isthat it is composed of multiple semi-independent unitpattern generators (or CPG subunits) that can be acti-vated or inactivated independently and functionallylinked in different temporal sequences. Thus, a numberof different motor patterns can be produced. In He-lisoma, there are three such subunits. During evolution,other gastropod species may have added an additionalinterneuronal subunit(s), or lost one, to accommodatedifferent behavioral niches.

Confirmation of cellular homologies is problematic(e.g. Croll, 1987; Breidbach and Kutsch, 1995). Here,we will not absorb ourselves with attempted ‘proofs’ ofhomology in specific cases. None-the-less, the Dar-winian paradigm of relation of species by commondescent is generally accepted at present. Analyses of celllineages and patterns of neuronal differentiation indi-cate clear homologies of identified neurons in somecases (cf. Comer and Robertson, this volume and in-cluded references). Many gastropod neurons have dis-tinguishable unique identities. Comparativemorphological, physiological, and neurochemical dataprovide a strong case for cellular homology for a fewgastropod neurons. Below, I will suggest candidatehomologies, especially for a number of CPG interneu-rons (cf. Fig. 14).

Perhaps the best evidence for homologies to date inmolluscan feeding circuitries relates to higher ordermodulatory interneurons and to giant buccal neuronsthat innervate the gut (e.g. Altrup, 1987; Lloyd et al.,1988; Bulloch and Ridgeway, 1995), or the salivaryglands (e.g. Kater et al., 1978; Bahls et al., 1980;Barber, 1983; Bahls et al., 1995). A pair of giantserotonergic neurons has been identified in the cerebralganglia of numerous gastropods and these neuronsdisplay great morphological, physiological, and func-tional similarities (e.g. Weiss and Kupfermann, 1976;Granzow and Rowell, 1981; Croll, 1987; Bulloch and

Ridgeway, 1995). More recently, neurons similar toHelisoma neuron Pl1, which inhibits buccal motor pat-terns, have been identified in a number of basomma-tophoran and stylomatophoran pulmonates and in thecarnivorous pteropod opisthobranch, Clione (Alania,1995; Alania and Sakharov, 1996 and M. Alania, per-sonal communication). It was suggested above thatneuron Pl1 was ideally situated to coordinate feedinglocomotion and defensive withdrawal. The effects ofPl1 on locomotion or withdrawal have yet to be exam-ined in Helisoma. However, increased activity in FMRF-amidergic pleurobuccal neurons in Clione, similar toHelisoma neurons Pl1, not only inhibited feeding butwas also correlated positively with both spontaneousand induced accelerations of the locomotor CPG (M.Alania, personal communication).

4.1. Biological and methodological complications forinterspecific comparisons of gastropod feeding

There are several real or potential factors that maycomplicate interspecific comparisons of gastropod feed-ing motor patterns and underlying neuronal circuitries.(1) Incomplete and non-congruent experimental datasets are available for each of the species that have beenexamined. (2) Interspecific diversity of feeding behav-iors in gastropds. (3) Intraspecific diversity of oralbehaviors and the underlying multiplicity of motorpatterns in any given species. (4) Incomplete congru-ence of action potential firing patterns in motor neu-rons with respect to those seen in the CPGinterneurons. Motor neuron firing patterns are morediverse and complex than are those in interneurons, dueto multiple interneuronal synaptic inputs, multiple ef-fects of transmitters, and diverse intrinsic membraneproperties among motor neurons, and the modulationof all of these. Thus, similar CPG patterns could ap-pear different purely as a function of the motor neuronsmonitored. All of the above factors can be addressed,as indicated below.

4.1.1. Incomplete dataOne major difficulty in revealing the generalities of

gastropod feeding has been that studies on differentspecies have focused on many different aspects of feed-ing. For instance, studies in the stylomatophora, and inthe carnivorous opisthobranch, Pleurobranchaea, havefocused primarily on sensory processing, behavioralchoice, higher order control of feeding, and its modula-tion by behavioral state or learning (e.g. London andGillette, 1984; Mpitsos and Cohan, 1986; Delaney andGelperin, 1990; Kemenes, 1994; Jing and Gillette,1995). Thus, relatively little is known about the organi-zation of the buccal CPGs in these species. Even inthose species in which the buccal CPGs have beenstudied extensively (e.g. Aplysia, Helisoma, Lymnaea,


Planorbis), different aspects of the CPGs have beenstudied. Concerted efforts at a comparative synthesis ofthe neuronal organization of feeding behaviors hasbeen lacking.

4.1.2. Interspecific di6ersityMechanisms of feeding in gastropods are diverse (e.g.

Audesirk and Audesirk, 1985; Thomas et al., 1985),leaving the question as to what extent neuronal cir-cuitry underlying these behaviors has been conservedphylogenetically. Perhaps the most disconcerting sce-nario in relation to a comparative synthesis could bethat gastropod feeding structures and behaviors mayhave diverged to such an extent that few commonalitiesremain in their buccal neuronal organization. This situ-ation may hold for some species with highly specializedfeeding, such as Melibe (Hurst, 1968) or Na6anax,(Susswein et al., 1987; Cappell et al., 1989a,b) where theneed for an odontophore has been eliminated largely orwholly. However, most gastropods that have been sub-jected to neurophysiological analyses use rhythmicmovements of a toothed radula during feeding. Theseinclude herbivorous or omnivorous pulmonates (e.g.the basommatophorans Helisoma, Planorbis andLymnaea and the stylomatophorans Achatina, Helix,Incilaria, Limax) and opisthobranchs that graze onalgae or sessile invertebrates (e.g. Aplysia, Tritonia).Even aggressive carnivorous opisthobranchs that usespecialized structures for capturing prey typically ingestor swallow the prey with rhythmic buccal movements(e.g. Clione, Pleurobranchaea).

Close phylogenetic relatedness, combined with simi-larities of ecological niches and feeding behaviors,should facilitate the analyses of potentially homologousneuronal organization. Although opisthobranchia andpulmonata represent different subclasses of gastropoda,a consensus suggests that they are much more closelyrelated than are the different subgroups of the highlydiverse ‘prosobranch subclass’, a paraphyletic groupingno longer considered systematically useful (cf. Ponderand Lindberg, 1996). Here, a significant degree of ho-mology of identifiable neurons of the feeding circuits ofopisthobranchs and pulmonates is hypothesized. Simi-larities in identified neurons and neuronal organizationare indicated below.

4.1.3. Intraspecific di6ersityA major biological factor obfuscating interspecific

comparisons of ‘feeding motor patterns’ and their neu-ral bases is that each species examined has a plasticmultifunctional CPG that generates multiple buccalmotor patterns. Some of these patterns (usually moni-tored with the nervous system in vitro or in a semi-in-tact preparation) mediate variations of feedingbehavior, some mediate regurgitation or other non-feeding oral behaviors and some of the patterns may

even be non-functional. Over the years, numerous buc-cal motor patterns have been asserted or implied in theliterature to represent fictive feeding, often on the basisof an electrophysiological recording from a single neu-ron and without concurrent behavioral data. Buccalmotor patterns often change qualitatively on a cycle bycycle basis and multiple qualitatively different motorpatterns from identified neurons have been depicted as‘fictive feeding’ in the same figure. Frequently, theoriginal author is conservative, referring to a recordingas a buccal motor pattern, only to have it referenced insubsequent reports as a ‘feeding motor pattern’. Howthen, amongst such confusion, can meaningful interspe-cific comparisons of feeding motor patterns and theirunderlying neuronal circuitry be made?

Fortunately, feeding behaviors and the dynamics ofbuccal masses and odontophores can be analyzed inintact freely behaving juvenile gastropods by videomi-croscopy. Reasonable facsimiles of feeding behaviorcan be evoked in semi-intact preparations while electro-physiological recordings are made from identifiableneurons and/or muscles (e.g. Arnett, 1996; Drushel etal., 1997). A ‘typical feeding behavior’ can be selectedand the standard feeding motor pattern underlying thisfeeding behavior determined. With effort and luck mo-tor neurons displaying common sets of PSPs and theinterneurons mediating these sets of PSPs can be iden-tified uniquely. Sets of phase-linked PSPs in multipleidentified motor neurons, suggestive of a common pre-synaptic interneuron or set of interneurons, may beseen to occur in linked fashion in temporal sequencesdifferent from that seen in the standard feeding pattern.Thus, plasticity in the organization of the buccal CPGcan be characterized, and sometimes controlled, phar-macologically or by modulatory interneurons. Finally,the interactions among identified modulatory interneu-rons, CPG interneurons and motor neurons can bestudied.

Key features of the organization of the Helisomabuccal CPG, and of patterns of activity recorded frombuccal motorneurons, help illustrate difficulties encoun-tered when comparing different motor patterns. Eachof the three CPG subunits is a semi-independent condi-tional oscillator that can generate rhythmic activity thatmay or may not be linked to activity in the othersubunits. Furthermore, the order of activity of particu-lar subunits is variable. For instance, subunit 3 maygenerate action potential bursts that are linked to S2activity but the S3 activity can precede that of S2,instead of following it as it does in the typical feedingbehavior (cf. Arnett, 1996; Quinlan and Murphy, 1996).A subunit also may be active more than once per cycle.For example, subunit 3 may generate bursts of activityboth preceding and following S2 activity (Fig. 12).Another complication relative to CPG analysis is thatthere are strong gradations in the intensity of S1 and S3


activity (cf. Quinlan and Murphy, 1996; Quinlan et al.,1997). Different interneurons active within the samephase of the feeding pattern can have different onsetsfor firing and not all interneurons of a given phase needto be active in all motor patterns.

4.1.4. Non-congruence of interneuronal and motorneuronal patterns

Perhaps the most prominent complication in feedingmotor pattern analysis across species arises from thefact that action potential burst patterns of many mo-toneurons will not show an abrupt onset or terminationof the bursts at the ‘borders’ between subunit activities,even if a rhythmic S1–S2–S3 standard feeding patternis being generated (see below). Therefore, some iden-tified motor neurons provide a much clearer reflectionof CPG activity than other identified motor neurons.To know for certain which motor neurons provide areliable monitor of CPG activity, relevant interneuronsmust be identified, and recorded with specific motorneurons under a variety of conditions (e.g. with differ-ent modulators in the bath). In general, more than oneneuron will need to be monitored simultaneously toobtain a complete reflection of CPG activity.

In Helisoma, since the onset and duration of burstsof action potentials in different S1 interneurons varies,there will be differences in firing onset in motor neuronsinnervated by the S1 interneurons. There is, however,an abrupt transition from S1 to S2 interneuronal activ-ity because S1 interneurons excite S2 interneurons tothreshold for plateau potential generation and S2 neu-

rons have feedback inhibition to terminate the S1bursts (Quinlan et al., 1995). In spite of this, manymotoneurons receive excitation both from S1 and fromS2 interneurons, and thus, may fire action potentialsacross phases 1 and 2 (or similarly 2 and 3, or even 1,2, and 3) of the feeding pattern (cf. Table 1). Anadditional complicating factor is that biphasic synapticresponses may occur in follower neurons. In particular,several neurons that are postsynaptic to S2 interneu-rons (e.g. neuron B18, which is primarily excited duringphase 3) show an initial hyperpolarization followed bya depolarization. A consequence of these neural organi-zational features is that while each ‘feeding’ motorneuron reflects the activity of one or more subunits ofthe buccal CPG, no single motor neuron provides adirect mirror image of the activity of the CPG.

One can overcome the above complications in motorpattern analysis by having unequivocally identifiableneurons that can serve as reference monitors of activityin CPG subunits. Helisoma neuron B19, for example, issuch a reference neuron that reflects reliably the activityof CPG subunits 2 and 3 (Quinlan and Murphy, 1996).One should monitor synaptic potentials and not justextracellular spiking patterns, because similar spikingpatterns can arise from different patterns of synapticactivity. Finally, interneurons must be identified andtheir activity confirmed to be coincident with CPGsubunit activity during feeding patterns. They must beshown to evoke phase-characteristic PSPs in appropri-ate identified follower neurons during feeding patterns.Hyperpolarization of the identified interneuron shouldeliminate at least some of the PSPs evoked by the CPGsubunit. Similarly, the identified interneuron should beable to evoke phase-characteristic PSPs in identifiedfollower neurons when stimulated in quiescent prepara-tions or in preparations generating non-feeding pat-terns. These PSPs need not be identical to the PSPsgenerated during feeding patterns since synaptic re-sponses may be modulated during feeding.

4.2. Specific buccal motor patterns

Triphasic patterns of motor neuron activity, similarto that shown above for Helisoma (Fig. 3) have beenreported to underlie feeding in the closely related pul-monate, Lymnaea (Elliott and Benjamin, 1985a) and inthe opisthobranch, Tritonia hombergi (Bulloch andDorsett, 1979). Somata of groups of neurons that areactive in equivalent phases of the feeding cycles inseveral different species have been localized to similarareas of their respective buccal ganglia (Bulloch andRidgeway, 1995).

Putative feeding cycles with two, three, or morephases have been reported in other gastropods. Biphas-ic cycles reported for the planorbid basommatophoransnails appear to represent partial characterizations of a

Fig. 12. Multiple patterns of inhibition and excitation in Helisomaneuron B19 indicate the flexibility of organization of the buccal CPG.During the typical S1–S2–S3 feeding motor pattern neuron B19 isinhibited by S2 neuron B2 and is excited by S3 neuron N3a. HoweverS2 and S3 are semi-independent interneuronal oscillators that can beindependently rhythmically active or linked in different temporalsequences. Upper left — rhythmic activity with S3 excitation preced-ing S2 inhibition. Upper right — rhythmic activity with S3 excitationboth preceding and following S2 inhibition. Lower left — rhythmicS2 inhibition with no S3 excitation. Lower right — rhythmic S3excitation with no S2 inhibition.


Fig. 13. A triphasic buccal motor pattern in Aplysia. A, Simultaneousintracellular recordings from a neuron B4/B5 displayed the character-istic ‘butterfly appearance’ that results from the transition from phase1 excitation to phase 2 excitation. An unidentified motorneuron(MN-1) displayed phase 1 inhibition, phase 2 excitation and phase 3inhibition. B, A neuron B8 displayed excitation in phases 1 and 2 ofa cycle while an unidentified motor neuron (MN-2) displayed distinc-tive inhibitory inputs during phases 1 and 2 followed by a phase 3excitation.

from phase 1 (protraction) innterneurons and fromPhase 2 (retraction) interneurons. The onset of thephase 2 depolarization corresponds to a decrease inaction potential amplitude and a resultant butterflyappearance of the recording (Fig. 13A cf. Hurwitz andSusswein, 1996). In the instance shown an unidentifiedmotor neuron of the ventral cluster displayed phase 1inhibition, phase 2 excitation, and inhibition in a thirdphase. This suggests that neurons B4/B5 may receiveexcitation from three different sets of interneurons.Similarly, neuron B8 is known to receive excitationduring both phase 1 and 2 (Morton and Chiel, 1993).Another unidentified ventral cluster motor neuron dis-played distinctive phase 1 and 2 IPSPs followed by aphase 3 burst of action potentials. These ‘phase 3effects’ could plausibly be explained by mechanisms notrequiring phase 3 interneurons, but they are consistentcertainly with and suggestive of a tripartite CPG. Itremains to be determined whether this third phase ofactivity in Aplysia arises from interneurons homologousto Helisoma interneuron N3a. Biphasic motor patternsin other gastropods such as Clione (Arshavsky et al.,1989), Pleurobranchea (Davis et al., 1973), and Helix(Peters and Altrup, 1984) too may represent partialcharacterizations of a triphasic pattern.

Buccal rhythms consisting of four or five phases havebeen reported and may result from the designation ofthe quiescent interval between cycles as a ‘phase’ of thecycle. For example, the triphasic pattern of feedingmotor neuron activity in Lymnaea (Elliott and Ben-jamin, 1985a) was earlier reported to be a four-phasecycle (cf. Fig. 14 in Benjamin and Rose, 1979). Alterna-tively, the retraction of the radula from its most pro-tracted position to a ‘rest’ position is sometimessubdivided into separate rasp and retraction phases.However, four or five phase cycles (cf. Fig. 2 in Sossinet al., 1987) are within the capabilities of a tripartitepattern generator with flexible linkages among indepen-dent conditional oscillators. For example, in Helisoma,a rhythmic four-phase S1–S3–S2–S3 pattern can begenerated (unpublished observations).

4.3. Interspecific comparisons of CPG interneurons andfeeding motor neurons

Few direct comparative studies of similar motor neu-rons and interneurons involved in feeding in differentgastropod species have been attempted. Though thebuccal ganglia of pulmonates and opisthobranchs havea plethora of ‘identifiable’ neurons, there are relativelyfew buccal neurons in any species that are truly‘uniquely identified.’ Insufficient physiological and mor-phological data is published to allow re-identification ofmost buccal neurons that at first glance would seem tobe ‘identified’. For instance, on the map of the caudal(aka dorsal) surface of the buccal ganglia of Helisoma

triphasic feeding cycle. The biphasic pattern of alternat-ing bursting in motor neurons described in Helisoma byKater (1974), as discussed above, corresponded clearlyto phases 2 and 3 of the standard feeding motorpattern. Similarly, Arshavsky et al. (1988a) described abiphasic pattern of protraction and retraction, sepa-rated by a quiescent interval, in Planorbis. The patterndescribed in Planorbis corresponded to phases 1 and 2of the Helisoma triphasic feeding pattern. Phase 3 mo-tor neurons that mediate the hyper-retraction of theradula inexplicably were not characterized in Planorbis,even though unlabeled somata of what appear to beneurons homologous to the large neurons B18 and B19of Helisoma were included in the Planorbis buccal map.In Aplysia, a biphasic pattern subserves protraction(phase 1) and retraction (phase 2) of the radula (Hur-witz et al., 1996). A third phase may be involved inswallowing. Perrins and Weiss (1998) presented atriphasic pattern in Aplysia but suggested that the thirdphase may actually represent a pause between biphasiccycles. However, triphasic buccal motor patterns resem-bling those of Helisoma are recorded readily in Aplysianeurons (Fig. 13). Neurons B4/B5 receive excitation


provided by Kater (1974) 23 pairs of neurons aredepicted and named. However, this study preceded theavailability of the dye, Lucifer Yellow and these neu-rons were ‘identified’ on the basis of relative soma sizeand position and patterns of electrophysiological activ-ity, with minimal morphological data from Procionstains. Only five of these 23 pairs of neurons areuniquely re-identifiable today (B4, B5, B17, B18, B19).With the exception of those five neurons plus B27, B101and B110, (Fig. 4) a combination of physiologicalrecordings and morphological stains is necessary forunique identification of Helisoma buccal neurons.

Simlarly, Benjamin and colleagues (Benjamin et al.,1979; Rose and Benjamin, 1979, 1981a,b) identified ten‘types’ of buccal follower neurons in Lymnaea withcriteria similar to those used by Kater (1974). Only fourof these ten types (neurons B1–B4) represent uniquelyidentifiable neurons without using morphological data,which has been provided only sporadically (cf. Staras etal., 1998). Arshavsky et al. (1988a,b,c) identified nine‘groups’ of buccal neurons in Planorbis, again usingsimilar physiological criteria to those of Kater and ofBenjamin, with only sporadic concurrent morphologicaldata. The current author is also not blameless. WhileLucifer Yellow is used routinely in microelectrodes,yielding morphological data concurrently with physio-logical data in this author’s laboratory, much of thatdata sits in reams of chart recordings and photographicnegatives on laboratory shelves. I used these examplesnot to castigate my colleagues but to illuminate the pastand current situations, and to call for more publishedevolutionarily significant data on identifiable neurons,both in print and on the world wide web (cf. Comerand Robertson, this volume).

In spite of the paucity of direct comparative studies,several analogous and potentially homologous neuronshave been identified in the basommatophoran snails,Helisoma, Planorbis and Lymnaea, and in two differentopisthobranchs, the sea hare, Aplysia, and thepteropod, Clione. The estimated number of neurons inthe buccal ganglia of these species varies by about afactor of two (Boyle et al., 1983). Thus, not everyneuron in one species will have a unique homolog inother species. For instance, a common developmentalcell lineage in two species might result in a lack of aone-to-one relationship between neurons if there is adifference in the number of cell divisions or in pro-grammed cell death in the two species. None-the-less itseems certain that these related gastropods have ho-mologous neurons in the sense that particular neuronsresult from similar developmental pathways resulting insimilar form and function (cf. Breidbach and Kutsch,1995). The comparisons below are made in an attemptto spur comparative research. More studies combiningmorphological, electrophysiological, pharmacological

and biochemical characterizations of ‘identified’ neu-rons are needed for each of the species investigated.

We focused on pure interneurons (i.e. no peripheralprocesses) and influential neurons (i.e. they participatein motor pattern expression but also have peripheralprocesses that may have sensory or motor functions).While many phase 1, 2, and 3 motor neurons have beenidentified in several species (e.g. Church and Lloyd,1994), many of these have great similarities to othermotor neurons in the same species and relatively fewhave been characterized sufficiently to allow ready in-terspecific comparisons. Neurons were characterized be-low on the basis of in which phase of the feeding cyclethey are most prominently active. Interneurons andinfluential neurons with major roles in the buccal CPGswere placed in a ‘composite universal tripartite CPGmodel’ (Fig. 14). Probable or potentially homologousinterneurons were boxed together.

Fig. 14. A composite model for a universal gastropod tripartitebuccal central pattern generator. The model displays three subunits(S1, S2, S3) of the CPG, each of which represents a semi-independentneuronal oscillator that provides the primary excitation to sets ofmotor neurons active during corresponding phases of a feeding cycle.Neurons in each of the large boxes are active during one of the threephases. Neurons within the smaller rectangles inside the large boxesrepresent putatively homologous interneurons based on morphologi-cal and physiological characteristics. In some cases, these putativehomologs have also been shown to have the same neurotransmitter.Note that the BCN1 group is a heterogenous set of phase 1 interneu-rons with apparent variability in number among the different gas-tropods. They have similar morphologies and are all phase 1interneurons but some fire early during phase 1 and some later duringphase. With the exception of Planorbis group 2, the other designa-tions represent individual identified neurons. The arrows indicate thatthere are excitatory interactions among interneurons of differenttypes within each subunit but should not suggest that every interneu-ron within a subunit excites every other interneuron. The bar with aperpendicular bar indicates that S1 interneurons excite S2 interneu-rons. The bars with filled circles represent inhibition. The darkerrectangles within each subunit represent the neurons that seem to benecessary and sufficient to generate a triphasic motor pattern. Thequestion marks indicate that, (1) a phase 1 interneuron with amorphology similar to Helisoma interneuron N1a was described inClione but not given a name; (2) the effects of Lymnaea interneuronsN3p and N3t are inhibitory to those motor neurons that are excitedby S3 so in that sense they are not a principal part of subunit 3; and(3) it is not yet known whether Aplysia B21 and B22 activatehypothetical phase 3 interneurons. However, like the Helisoma clusterof influential neurons B101–104, they are small cardioactive peptideimmunoreactive radular mechanoafferent neurons on the rostral sur-faces of the buccal ganglia. They have similar morphologies and theyfire action potentials during the latter part of spontaneous buccalmotor patterns (i.e. during phase 3). H=Helisoma ; P=Planorbis ;L=Lymnaea ; A=Aplysia ; C=Clione.


Table 2Analogous, possibly homologous motor neurons and influential neurons in Helisoma (H.), Lymnaea (L.), and Aplysia (A.)a

Aplysia Axonal projections Muscle targetsHelisoma Lymnaea

Phase 1 (protraction) neuronsIn, B31/32 H. PBNs=A. RN H. pj m=A. I2 mIn, B6B61/62 H. PBN, A. RN, L. pjNB7nor H. and L. pj=A.I2 mB8A. SRT mn H. VBN=A. N1; H.?, A. SRT, L. pj?B7 B7a

L. VBN or LBN?B3 B5 H. and L. DBN ?

Phase 2 (retraction) neuronsH. and L. PBNsIn, B29 ? Perhaps rad tens mIn, B10

Phase 3 (hyper-retraction) neuronsB19 B4 H. and L. VBNs and LBNs H. and L. slrt and aj

a in, Influential; PBN, posterobuccal nerve; RN, radular nerve, PJ, posterior jugalis; nor, nitric oxide responsive; SRT, subradular tissue; VBN,ventrobuccal nerve; LBN, laterobuccal nerve; DBN, dorsobuccal nerve; rad tens, radular tensor; slrt, supralateral radular tensor; aj, anteriorjugalis.

The distinction between influential and motor neu-rons is somewhat arbitrary. There are gradations in theabilities of motor neurons to influence the buccalCPGs, in large part because of extensive electrotoniccoupling not only within but across these groups. It haslong been known that molluscan motor neurons caninfluence interneurons. For instance, it was shown byGranzow (1979) that motor neuron B19 could, in somepreparations, influence the buccal CPG. Some poten-tially homologous motor neurons and influential neu-rons that were not placed in the composite CPG modelare indicated in Table 2.

4.4. Protraction phase interneurons

Very similar, perhaps homologous, phase 1 (protrac-tion) catecholaminergic (almost certainly dopaminergic)interneurons have been characterized in Helisoma(Quinlan et al., 1997) and Aplysia (Kabotyanski et al.,1998). Dopamine may be a universal initiator of gas-tropod feeding motor patterns (e.g. Wieland andGelperin, 1983; Kyriakides and McCrohan, 1989;Kabotyanski et al., 1994; Quinlan et al., 1997). NeuronsN1a in Helisoma and B65 in Aplysia are similar strik-ingly. Their somata are in similar locations near thelateral edges of the caudal surfaces of the buccal gan-glia. The main axons cross the buccal commissure butdo not exit the buccal ganglia. They have neuriticarbors in each of the paired buccal ganglia. Stimulationof these neurons mimics the effects of bath applicationof dopamine or DOPA. Both neurons N1a and B65displayed catecholamine histochemical staining. In He-lisoma, the dopamine antagonist, sulpiride, blocked theeffects of bath application of dopamine and of neuronN1a stimulation.

Lymnaea interneuron N1L is also a candidate ho-molog of Helisoma neuron N1a and Aplysia neuronB65 and is similar to them in relative soma position,

morphology and physiological effects on buccal motorpatterns (Yeoman et al., 1995). It has been reported tobe non-dopaminergic but the negative evidence is notcompelling (Vehovsky and Elliott, 1995). The dopamin-ergic antagonists fluphenazine and ergometrine failed toblock the effects of stimulation of Lymnaea interneuronN1L. However, the effectiveness of neither antagonistagainst exogenous dopamine was tested in this system.Furthermore, somata in the vicinity of that of Lymnaeaneuron N1L displayed dopamine immunoreactivity(Elekes et al., 1991). Another possible homolog of theseneurons is a protraction phase interneuron, which has asimilar morphology and soma position in the carnivo-rous opisthobranch, Clione (Arshavsky et al., 1989).

Two other types of phase 1 pure buccal interneuronsin Helisoma, neurons N1b and N1c, were describedabove (Fig. 8). Neuron N1b is morphologically, at leastsuperficially, similar to the dopaminergic Aplysia in-terneuron B20 (Teyke et al., 1993). There are somatathat display catecholaminergic histochemistry in thevicinity of the somata of Helisoma interneurons N1b,but whether these interneurons are dopaminergic re-mains to be determined. Interneurons N1c have somatalocated laterally on the dorsal surfaces of the buccalganglia. Interneuron N1c projects an axon to the con-tralateral buccal ganglion that forms a loop and returnsto the ipsilateral buccal ganglion (Fig. 8). This protrac-tion phase interneuron is putatively a homolog of theSO interneuron of Lymnaea studied extensively (Elliottand Benjamin, 1985b; Yeoman et al., 1993). A similarphase 1 interneuron, also designated the SO, was alsoreported in Planorbis (Arshavsky et al., 1988a,c). In-terneurons of this type have, thus, far been describedonly in basommatophoran snails.

A widespread class of protraction phase interneuronsare represented by the type 1 bucco-cerebral interneu-rons (BCN1 neurons) in Helisoma. Depolarization ofthese neurons can drive feeding motor patterns and


their action potentials can evoke one-for-one EPSPs insome protraction phase motor neurons. Neurons of thisclass have somata near the margins of, or sometimesunderneath, the somata of the medial giant buccalneurons (e.g. Helisoma neuron B5, Lymnaea neuronB2) that innervate the esophagi of Helisoma, Planorbis,Lymnaea, Aplysia, and Clione (Arshavsky et al., 1988a,1989; Richmond et al., 1991). The BCN1 interneuronsproject an axon across the buccal commissure to tra-verse the contralateral cerebrobuccal connective andenter the cerebral ganglion. Each has a neuritic arbor inboth buccal ganglia and a terminal arbor in the cerebralganglion contralateral to the BCN1 soma. Retrogradestains of severed CBCs in Helisoma with either CoCl2or Lucifer Yellow show consistently the three neuronsof this morphological type in each buccal ganglion. Thethree pairs of BCN1 neurons are not yet readily distin-guishable from each other, but they are heterogenous.Two of the three pairs of BCN1 neurons in Helisomaare GABA-immunoreactive (Richmond et al., 1991).

The numbers of this class of phase 1 interneuronsappear to be different in different gastropods. Planor-bis, (family Planorbidae, like Helisoma) was reported tohave four pairs of these neurons, based on retrogradebackfills of the CBC (Arshavsky et al., 1988a). Origi-nally, based upon physiological recordings and a fewLucifer Yellow injections, there were thought to beabout ten pairs of interneurons of this type in Lymnaea(Rose and Benjamin, 1981b; Elliott and Benjamin,1985a). They were referred to at that time as N1interneurons. It now appears, based upon retrogradestaining of CBCs, that there is only one pair of neuronsof this type in Lymnaea (Kemenes and Elliott, 1994). Itis called the medial N1 neuron or neuron N1M(Yeoman et al., 1995).

Two protraction phase interneurons, B34 and B63,similar to the Helisoma BCN1 morphological type,have been identified in the opisthobranch Aplysia (Hur-witz et al., 1997). Interestingly, interneuron B63 ap-pears to be active during the protraction phase of allbuccal motor patterns that have a protraction phase,whereas B34 appears to be devoted to egestion patterns.The carnivorous opisthobranch, Clione, based uponretrograde staining of the CBC, is thought to have onlya single pair of protraction phase interneurons (PIN1)of this morphological type (Arshavsky et al., 1989).Thus, currently, there appears to be a greater numberof BCN1 type phase 1 interneurons in the pulmonatebasommatophoran planorbid snails than in either theclosely related pulmonate basommatophoran lymnaeidsnails or the opisthobranchs. However, these relativenumbers are based on examinations of very few species,and are based largely upon retrograde staining tech-niques, which can be quite capricious.

4.5. Motor and influential neurons

Helisoma phase 1 motor neurons B3, B6, B7, and B8were described above (Fig. 5). Motor neurons B31,B32, B61 and B62 of Aplysia have physiological andmorphological similarities to neurons B6 and B8 ofHelisoma. Aplysia motor neurons B31 and B32 andB61/B62 innervate the intrinsic-2 (I2) muscle, a protrac-tor muscle that is analogous/homologous to the pjmuscle innervated by B6 and B8 of Helisoma (Hurwitzet al., 1994; Arnett, 1996). Neurons B31/B32 of Aplysiaand B6 of Helisoma are strongly influential and canevoke buccal motor patterns. An SRT (subradular tis-sue) motor neuron in Aplysia appears morphologicallyand physiologically similar to Helisoma neuron B7 (Fig.5 and Borovikov et al., 1997), but it is not yet known ifHelisoma’s B7 innervates the SRT. These neurons haveaxons in equivalent buccal nerves (Aplysia N3 andHelisoma VBN, formerly called the HBN). Two phase 1motor neurons were uniquely identified recently inLymnaea, B7a and B7nor. Neuron B7a is somewhatsimilar morphologically to Helisoma B7 and Aplysia’sSRT motor neuron in that it has an axon in either theipslateral VBN or the LBN (these nerves were notdistinguished). Neuron B7a is also an influential neuronthat is electrotonically coupled to neuron N1M (Staraset al., 1998). Lymnaea neuron B7nor is responsive tonitric oxide and innervates the pj muscle via the pjnerve (Park et al., 1998), and may be related to theHelisoma B6, B8 and Aplysia B31/B32, B61/B62neurons.

4.6. Retraction phase interneurons

Pure phase 2 buccal interneurons with no axonstraversing buccal nerve roots include the group 2 neu-rons of Planorbis, and neuron B64 in Aplysia (Ar-shavsky et al., 1988b; Hurwitz and Susswein, 1996).The Planorbis group 2 neurons displayed endogenousrhythmic activity when isolated from the buccal ganglia(Arshavsky et al., 1988b). Aplysia interneuron B64 wasfound to be active consistently during Aplysia buccalmotor patterns. However, hyperpolarization of left andright neurons B64 failed to eliminate phase 2 PSPs andfailed to disrupt buccal motor patterns. Such hyperpo-larization revealed large phase 2 depolarizations resem-bling the plateau potentials seen in the glutamatergicinfluential interneurons B2 of Helisoma and N2v ofLymnaea (see below). It appears that Aplysia neuronB64 and the Planorbis group 2 neurons may contributeto CPG subunit 2 rhythmicity and have synaptic effectson subsets of buccal interneurons, but activity in theinfluential glutamatergic interneurons like B2 of He-lisoma and N2v of Lymnaea may be necessary andsufficient to provide the primary output of subunit 2.


Glutamate is the primary phase 2 (retraction phase)neurotransmitter, evoking pharmacologically distinctEPSPs and IPSPs in subsets of buccal motor neurons inHelisoma (Quinlan and Murphy, 1991). Stimulation ofneuron B2 by current injection evokes phase 2-likePSPs in all buccal neurons examined. Hyperpolariza-tion of interneuron B2 during ongoing buccal motorpatterns eliminates phase 2 PSPs in all buccal neuronsexamined (Quinlan et al., 1995). Interneuron B2 ex-tends an axon to the contralateral buccal ganglion,where it makes a loop and extends back to the ipsilat-eral buccal ganglion. It forms a neuritic arbor in bothbuccal ganglia and it extends at least one, sometimestwo axonal branches out each of the posterior buccalnerves.

As with Helisoma, glutamate is the primary phase 2neurotransmitter in Lymnaea. Recently, Brierly andcolleagues (Brierley et al., 1997a,b,c) described a virtu-ally identical and presumably homologous retractionphase, presumptively glutamatergic, interneuron (N2v)in Lymnaea. The originally identified Lymnaea retrac-tion phase N2 interneurons (now called N2d) wereshown by photoablation to be unnecessary for theproduction of normal buccal motor patterns (Rose andBenjamin, 1981b; Kemenes and Elliott, 1994). The N2dneurons have peripheral axons and may be primarilysensory or motor neurons that are coupled electrotoni-cally to interneuron N2v. Neurons B29 in Helisoma(Figs. 6 and 10) and B10 in Lymnaea (Staras et al.,1998) are morphologically similar, relatively weakly,influential phase 2 neurons. Putative glutamatergic re-traction phase interneurons in Aplysia remain to beidentified but glutamate mimics some of the retractionphase PSPs in Aplysia (Fig. 15).

4.7. Hyper-retraction phase interneurons

Comparisons of phase 3 interneurons among gas-tropods are more difficult than comparisons amongphase 1 and phase 2 interneurons because of ‘pre-sumably missing data’. In Helisoma, interneurons N3a(Fig. 10) account for the primary excitatory and in-hibitory phase 3 PSPs in buccal motor neurons. Stimu-lation of interneuron N3a evokes phase 3-like PSPs in

quiescent preparations, and inhibition of interneuronsN3a eliminates phase 3 PSPs in buccal motor patterns(Quinlan and Murphy, 1996). The ‘phase 3 interneu-rons’ of Lymnaea clearly are neither morphologicallynor functionally equivalent to interneuron N3a of He-lisoma. Two types of phase 3 interneurons, N3 tonic(N3t), and N3 phasic (N3p) have been identified inLymnaea. If one hypothesizes that both Helisoma andLymnaea have homologs of each of these phase 3interneuronal types the relationships among the realand hypothetical neuronal types remain unclear. Effectsof eliminating activity in either N3t or N3p during afeeding buccal motor pattern in Lymnaea have not beendemonstrated. Both neurons evoke PSPs on buccalinterneurons and motor neurons. However, it is partic-ularly unclear why neuron N3t is considered part of thebuccal CPG. Interneuron N3t fires tonic action poten-tials essentially continuously except when it is inhibitedby phase 1 or 2 interneurons, hence the appellation‘tonic’. It is active in quiescent ganglia, and it is activein the ‘inactive interval’ between cycles during slowbuccal rhythmic activity. Thus, the PSPs evoked by N3tvery much resemble the tonic chloride-dependent PSPsrecorded in many Helisoma buccal neurons (e.g. B19)either during, or in the absence of, buccal motor pat-terns (Kater, 1974). The Lymnaea N3p neuronal activ-ity appears restricted to phase 3 of the Lymnaea feedingpattern. However, its synaptic effects are opposite insign of those of Helisoma phase 3 interneuron N3a onequivalent classes of motor neurons. For instance, thereis a set of similar hyper-retraction (aka swallowing) andradular tensor motor neurons in Helisoma andLymnaea. The largest of these motor neurons are B19in Helisoma and B4 in Lymnaea. These neurons havesimilar positions of their somata, similar morphologies,similar (though not identical) physiological activity (seesection on comparisons of motor neurons below), andsimilar muscular targets. Both neurons innervate theslrt and aj muscles. Helisoma neuron B19 is excited byphase 3 interneuron N3a. In fact, the firing patterns ofB19 and of interneuron N3a are almost identical toeach other during feeding and other motor patterns(Quinlan and Murphy, 1996). The Lymnaea N3p in-terneuron, however, evokes IPSPs on Lymnaea radular

Fig. 15. Glutamate depolarizes at least some Aplysia buccal neurons that are excited during the retraction phase of the feeding cycle. Anintracellular recording from an Aplysia neuron B4/B5 showed no spiking activity in Aplysia saline. Glutamate (1 mM) was superfused over thepreparation (first arrowhead) and it caused a depolarization of about 10 mV, which resulted in continuous firing of action potentials. Note thatthe glutamate also triggered some EPSPs onto neuron B4/B5 as indicated by the upward deflection of the baseline and a concomitant decreasein action potential amplitude. At the second arrowhead, normal physiological saline (NS) was superfused over the preparation.


tensor neuron B4 during the action potential burst ofneuron B4. Thus, for these tensor motor neurons, in-terneuron N3p (and N3t) appears to act as a brake onthe system. Clearly, they do not drive the action poten-tial bursts that occur in phase 3 motor neurons (e.g.B4). To quote Rose and Benjamin (1981a)), ‘‘The inputwhich we have called ‘N3’ is more interesting. Althoughit is difficult to see what function this input performs ininhibiting cells which are already hyperpolarized (5cells, 7 cells) it is clear that the N3 inhibitory inputfractionates 4-group and 8 cell bursts into a series ofshort duration bursts …. It is difficult to provide afunctional explanation of the excitatory N3 input to the3 cell, since this occurs after the 3 cell burst, and all thee.p.s.p.s are subthreshold’’.

The phase 3 bursts in Lymnaea B4 (and four-clustercells) have been attributed to post-inhibitory rebound(PIR) in the motor neurons, just as phase 3 activity inHelisoma B19 was attributed originally to PIR (Kater,1974). However, Lymnaea’s neuron B4 shows activitypatterns similar to Helisoma ’s B19 including rhythmicand sporadic excitation independent of phase 2 inhibi-tion. This is consistent with the presence of an uniden-tified excitatory interneuron similar to interneuron N3a,which accounts for the primary phase 3 excitation inHelisoma motor neurons (compare Figs. 12 and 16).

There is evidence for phase 3 excitation and inhibi-tion in Aplysia (Fig. 13). However, no Aplysia interneu-rons similar either to Helisoma neuron N3a or Lymnaeaneurons N3t and N3p have been reported. However,Aplysia has a population of SCP-immunoreactive radu-lar mechanoafferents on the rostral surface of the buc-cal ganglia. These neurons are morphologically similarto the B101–104 (akaVB1–VB4) cluster of HelisomaSCPB-ir radular mechanoafferents. These Helisomaneurons are effectively phase 3 interneurons (seeabove). In addition, at least some of the Aplysia radularmechanoafferents fire action potentials in the latter part(swallowing phase?) of spontaneous buccal motor pat-terns (Miller et al., 1994). It remains to be seen whetherstimulation of these Aplysia neurons can evoke PSPs,similar to swallowing phase PSPs in buccal motorneurons. Direct comparative studies of hyperretraction/swallowing phase interneurons are especially needed atthis time.

5. Concluding remarks

We have entered the fourth decade of research onidentified neurons and the circuits which they comprise.It is appropriate to consider what they have taught usand what they might teach us in the near future.Perhaps the single greatest point of consensus to ariseduring this period is that what were initially considered‘simple systems’ are not simple. Though not simple,

Fig. 16. Multiple patterns of activity in Lymnaea mimic the motorplasticity seen in Helisoma. During a typical feeding pattern (uppertrace) Lymnaea neuron B4 receives inhibition during phase 2 andexcitation during phase 3. However, phase 3-like excitation and phase2-like inhibition can be linked to produce patterns other than thestandard feeding pattern such as the S3–S2–S3 pattern (secondtrace). Each subunit can also be active independently yielding eithera rhythmic S2 pattern (third trace) or a rhythmic S3 pattern (lowertrace). All four of these patterns were recorded from the same neuronB4. The similarity of motor patterns suggests a similar CPG organiza-tion to that seen in Helisoma.

many invertebrate systems have the great attribute ofexperimental tractability. This experimental tractabilityof systems with relatively small numbers of identifiableneurons led to another major finding — in general,hard-wired circuit diagrams do not underly particularbehaviors. There has been a demise of the concept of‘stereotypy’ with respect to behaviors and the neuraloutputs of even ‘relatively simple’ circuits. The well-modulated motor system emerged triumphant. Withoutthe identifiability of unique neurons, it would be virtu-ally impossible to ascertain the degree, to which a singleneuron’s membrane characteristics and functional inter-actions with other neurons could be modulated to fitdifferent situations at different times.

It has also become clear that similar rhythmic motorprograms — that is, activity in two or more sets of


motorneurons with bursts of action potentials offset byphase lags — could arise from a wide variety of neuralcircuits. However, the characterized circuits were allcomposed using a circumscribed number of discrete‘building blocks’, intrinsic membrane properties andtypes of synapses (cf. Getting, 1989). Both intrinsicmembrane properties and specific synaptic connectionscan be modulated on a moment to moment basis sothat multiple functional motor patterns can arise froma given group of identified neurons. As indicatedthroughout this volume, future progress based uponidentified neurons will encompass further reductionistand molecular analyses of CPG building blocks andcomputational analyses of circuit function.

During the past 25 years, some of the experimentaldisadvantages of small mammalian neurons versuslarge identifiable invertebrate neurons have been miti-gated by technical advances — notably the widespreaddevelopment of patch clamp techniques and molecularexpression systems. Many laboratories working initiallywith the systems of identified neurons in invertebrateshave switched to mammalian systems. In addition, thediversity of circuits with similar outputs, the common-ality of circuit components, and the functional modula-tion of a given circuit to produce diverse outputs, haveled to the suggestion that what we have already learnedfrom several invertebrate systems may have effectivelybrought to an end ‘the search for new CPG circuits’.We feel that suggestion to represent a short-sightedview. Though the technical advantages offered by largeidentifiable neurons over smaller neurons lacking aunique identity may have decreased, the greatest at-tributes of identifiable neurons for neurobiologists aretheir unique identities per se! These unique identitiesallow short- and long-term analyses of circuit functionsand their modulations. Furthermore, ‘‘With this com-pendium of data, we will have the unique opportunityto examine the interspecific diversity of the neuronalmechanisms underlying a specific behavior. This ap-proach will be the reductionistic counterpart to theethological analysis of phylogenetic ritualization(Lorenz, 1966) and should provide significant insightinto the evolution of neuronal circuitry.’’ This opti-mistic quotation marks the closing remarks of Kater(1974). We are now poised to realize this opportunity.

Acknowledgements

Work reported here was supported in part by NSFgrants BNS-9121374 and IBN-9728769 and by the Uni-versity of Illinois Campus Research Board. I thankBridgette Arnett for the use of figures from her Ph.D.thesis and Danielle Turley for the data in Fig. 16. Ithank Jack Gibbons, Andy Carol, John Dowd andMike Hollinstat for helping in figure preparations and

Fred Bahls, Kelly Karas-Hedrick, Irene Yashina andVicky Leung for reading earlier versions of themanuscript.

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