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date: 24 February 2020

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Summary and Keywords

It is conceptually reasonable to explore how the evolution of behavior involves changes in neural circuitry. Progress in determining this evolutionary relationship has been limited in neuroscience because of difficulties in identifying individual neurons that contribute to the evolutionary development of behaviors across species. However, the results from the feeding systems of gastropod mollusks provide evidence for this concept of co-evolution because the evolution of different types of feeding behaviors in this diverse group of mollusks is mirrored by species-specific changes in neural circuitry. The evolution of feeding behaviors involves changes in the motor actions that allow diverse food items to be acquired and ingested. The evolution in neural control accompanies this variation in food and the associated changes in flexibility of feeding behaviors. This is present in components of the feeding network that are involved in decision making, rhythm generation, and behavioral switching but is absent in background mechanisms that are conserved across species, such as those controlling arousal state. These findings show how evolutionary changes, even at the single neuron level, closely reflect the details of behavioral evolution.

Keywords: neural circuits, evolution, homology, gastropods, feeding behavior, synaptic connections, central pattern generators, modulation, hunger and satiety, multitasking


On account of the extraordinary and in some cases bizarre feeding specializations of gastropod mollusks, there has been a continuing interest in the evolution of the behavioral and neural mechanisms underlying their food gathering and food processing mechanisms. Gastropod nervous systems have been particularly important in the research area that relates neural circuit evolution to behavioral evolution because they contain individually identifiable neurons allowing neural circuits underlying related behaviors to be compared across species. How neural circuit evolution relates to behavioral evolution is not well understood in neuroscience (Newcomb et al., 2012), and research on gastropod behavior is of particular interest in investigating these matters because more is known about motor circuits in categorically related behaviors than any other phylogenetic group (Sakurai & Katz, 2015).

To discuss the evolution and comparative studies of gastropod feeding, it is necessary to outline the advances revealed by molecular studies on the phylogeny of gastropod mollusks (Kocot, Halanych, & Krug, 2013; Waegele et al., 2014; Wanninger & Wollesen, 2018). These have led to a major revision of the taxonomy and phylogeny of the species described in this article (phylogenetic tree, Figure 1A). All of these species belong to one phylogenetic clade, the Heterobranchia and more specifically the subclade Euthyneura (Sakurai & Katz, 2015). The common ancestor of the Euthyneura was herbivorous whereas the carnivorous gastropods evolved several times (Goebbeler & Klussmann-Kolb, 2011). It is possible that the stereotyped kind of grazing behavior shown by large numbers of the panpulmonate molluskan species evolved from an earlier herbivorous ancestor whereas the multiple types of feeding behaviors shown by the carnivorous Nudibranchia and Pteropoda result from speciation resulting from the much greater variety of the animal prey that were available in their environment. Nevertheless, despite the variety of gastropod subclades used for neurobiological studies, the widespread presence of homologous neurons and muscle types in these diverse behavioral types provides the opportunity for circuit evolution to be related to behavioral evolution.

The organization of gastropod feeding networks into functional units (Benjamin, 2012) allows comparison of circuit mechanisms in gastropods with the same or different feeding behaviors. Specific neurons contribute to different network functions such as pattern generation (central pattern generators, CPGs), initiation (decision making), modulation (extrinsic and intrinsic), internal or motivational state (hunger and satiety and arousal) and multitasking (egestion and ingestion). The presence of these functional units consisting of individually identifiable neurons help in the process of targeting the particular network functions that are modified as part of the evolutionary process. The evolution of feeding behaviors involves changes in the motor actions that allow diverse food items to be acquired and ingested. How the circuit and muscular motor control systems evolved to allow the consumption of these various types of food is the major question. In addition, it is necessary to know whether neuronal mechanisms involved in behavioral state functions such as hunger and satiety and arousal also change as part of the evolutionary process.

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Figure 1. A comparison of phylogenetic, central ganglia organization and food acquisition mechanisms in a variety of gastropod mollusks (A) Phylogenetic tree of gastropod species (species images shown below) used for the evolutionary analysis of feeding (image of Pleurobranchaea from Noboa & Gillette, 2013). (B) The organization of the central ganglia in four of these species. (C) The central ganglia (CNS, central nervous system) of snails (e.g. Lymnaea) are concentrated around the gut and on the buccal mass in the anterior head part of the animal body. The feeding circuitry is in the paired buccal and cerebral ganglia. (D) The anterior head region of Lymnaea. Chemosensory cells are present on the lips and tentacles (tent.). The mouth and lips are shown (left side image). When grazing the mouth opens and the radula is protracted through the mouth (right side image). The mandibles (mand.) are important in the alternative biting behavior of the snail (modified from Crossley et al., 2016). (E) The locations of the lips, rhinophores (rh) and mouth in the herbivorous euopisthobranch, Aplysia (left image). During grasping behavior the “open” radula is protracted through the mouth (right image) ready to grasp stem-like food items (modified from Nargeot & Simmers, 2012). (F) The carnivorous nudipleuran species Pleurobranchaea uses the rhinophores and the oral veil to detect prey, the oral veil (ov) is pressed down on the prey when contact is made and the proboscis extruded to acquire the prey (left image). The radula is protracted through the mouth in a bite-strike sequence (radula shown “open” in the central image) (modified from Noboa & Gillette, 2013). (G) In the swimming carnivorous euopisthobranch Clione, the tentacle-like buccal cones (bc) grab the prey (a smaller pteropods) and hold it next to the mouth. The hooks (hk) extract the soft tissue from the prey’s shell (modified from Latorre et al., 2013).

The Variety of Feeding Behaviors

Gastropod mollusks of neurobiological interest show a striking variety of feeding behaviors from the continuous grazing activity of herbivorous panpulmonates to the sudden prey capture of carnivorous euopisthobranchs. The neural networks that control these diverse behaviors are located in the buccal and cerebral ganglia of the central nervous system (Figure 1B) adjacent to the feeding apparatus (buccal mass, Figure 1C). Feeding consists of a sequence of appetitive and consummatory behaviors that evolved in accordance with the type of food consumed and its location in the environment. Appetitive foraging behavior seeks to find food and orientates the animal toward the food source. It involves locomotion and body movements that locate the food close to the mouth. Then the consummatory behavior utilizes the feeding apparatus to ingest the food.

Herbivorous Gastropods

Panpulmonate pond snails such as Lymnaea and Biomphalaria have appetitive behaviors that allow the animals to localize food stimuli from a distance in still water using tropotaxis to orientate toward food (Fraenkel & Gunn, 1961). Simultaneous comparisons of chemical signals at symmetrical paired receptor sites result in a turn directly toward the more strongly stimulated side. Closer to the food where stronger concentration gradients are present, zig-zag motions are observed (e.g., Townsend, 1973). This is klinotaxis, a form of orientation where the snails compare the chemical intensity at successive symmetrical points in space. Spatial sampling of the type required for distance chemical orientation involves the pair of bilaterally symmetrical tentacles located on the left and right side of the head (Townsend, 1974; Wyeth & Croll, 2011). Pond snails often live in slow-flowing streams and rivers and locate food by moving upstream toward chemical signals (positive rheotaxis), another mechanism available for locating distant food sources (Kemenes & Benjamin, 1989). Appetitive behavior also includes “spontaneous” feeding movements, an exploratory foraging mechanism that allows the environment to be randomly sampled for the presence of food (Tuersley, 1989).

The consummatory behavior of panpulmonate pond snails such as Lymnaea, Helisoma and Planorbarius consists of bouts of continuous cyclical programs of rasps often lasting for several hundred cycles using a “tongue” or toothed radula that allows the snail to ingest the uniform algal film on which they often feed. This grazing type of feeding, on an algal substratum, often occurs during forward locomotion and the side-to-side head movements lead to a characteristic zig-zag feeding “trail” (Dawkins, 1974) whose role is to increase the efficiency of feeding by expanding the area of the food substrate available for consumption. In addition, Lymnaea and Helisoma cut small pieces of leaf from the edges of floating pond-weed by sweeping the lip of the odontophore past the dorsal mandibles (Figure 1D) in a scissorlike movement (Benjamin, 1983; Murphy, 2001). Terrestrial snails such as Limax also show similar jaw-mediated biting movements (Reingold & Gelperin, 1980) following eversion of the lips and their application to solid food (Gelperin, Chang, & Reingold, 1978). Although grazing behavior on a uniform algal substratum results from stereotyped cyclical rasping movements of the radula, on more variable food substrates the motor program responds to differences in the physical properties of the food. For instance, snails show a higher “bite cycle” frequency on soft food compared with hard food (Reingold & Gelperin, 1980; Large et al., 2006).

The euopisthobranch mollusk, Aplysia californica, is a principally herbivorous species, but it shows more flexible feeding behavior than panpulmonates such as Lymnaea. For example, its appetitive behavior is quite variable. Introduction of its preferred food, the red seaweed, Laurencia (Kupfermann & Carew, 1974), near a quiescent animal results in a sequence of appetitive behaviors (Kupfermann, 1974). An initial response consists of small movements of the rhinophores and tentacles followed by head waving and lifting of the anterior of the body to adopt a characteristic feeding posture that includes the movement of its head and neck back and forth. If after a period of head waving the animal does not come into contact with food, it lowers its body to the substrate and resumes locomotion and head waving until food is contacted.

Application of food stimuli to the region of the skin around the mouth triggers the consummatory phase of feeding. A series of flexible biting radula movements are initiated (Figure 1E) that function to grasp and tear off a piece of seaweed and bring it into the buccal cavity (Kupfermann, 1974). The initial bite phase is followed by a swallow (bite-swallow sequence) when the food enters the oral cavity. Food items of small size that are palatable reach the esophagus by a second phase of swallowing. Consumption of large pieces of seaweed cause an initial bite followed by a number of swallowing movements that progressively draw the seaweed into the buccal cavity.

Grazing is observed in Aplysia under field conditions (Kupfermann & Carew, 1974). Thus, Aplysia has two types of feeding, grasping, and grazing, dependent on the type of food. However, there is no evidence that Aplysia carries out spontaneous appetitive grazing movements that in Lymnaea sample the environment for the presence of food.

Carnivorous Gastropods

Pleurobranchaea californica is a generalist carnivore, a great predator. Appetitive responses occur in response to prey or extracts of prey. The animal turns toward the food source and carries out searching movements with its extended proboscis (Figure 1F) (Davis & Mpitsos, 1971). Contacts with potential food items such as squid elicit a consummatory response consisting of the rapid eversion of the proboscis through the open mouth and an explosive bite-strike movement by the radula. The radula movements act to grasp the food and gradually pull it into the mouth and buccal cavity by a series of rhythmic ingestion movements to be then swallowed into the gut. The prey are also squirted with paralyzing H2SO4 when in the buccal cavity (Morse, 1984).

The appetitive phase of feeding behavior in Navanax consists of the tracking of a mucus trail laid down by other smaller species of euopisthobranchs (e.g., Bulla sp.) that are its prey (Paine, 1963). The consummatory phase of feeding is triggered by contact with prey; Navanax rapidly sucks in the whole animal through the open mouth (Figure 2D) (Spira, Spray, & Bennett, 1980). Seizure of the prey is extremely rapid, less than half a second in duration (Susswein et al., 1984). The details of the ingestion sequence are highly flexible reflecting the variability in the size and compressibility of the food consumed (Susswein et al., 1984). When Navanax is presented with food too large to swallow, they either cease to respond to food after a few presentations or maintain suction on the partially swallowed prey (Susswein & Bennett, 1979).

The pteropod mollusk, Clione limacina, is a marine swimming gastropod that has unique prehensile appendages for prey capture and food ingestion, which distinguishes it from other gastropod mollusks (Figure 1G). It is a highly specialized carnivore that feeds on two other species of shelled pteropod mollusks of the genus Limacina. During steady swimming it orientates vertically with head up and maintains itself at a constant depth due to continuous up-and-down movements of lateral “wings,” waiting for its prey to approach. A sequence of appetitive behaviors initiated by the detection of prey by chemoreception lead to rapid forward swimming toward the food followed by prey capture once contact is made. The tentacle-like oral appendages, the buccal cones, protract by hydraulic means to surround the Limacina shell and hold it during the subsequent consummatory phases of feeding. After prey capture, the buccal cones manipulate the Limacina shell so that its aperture presses against the mouth of Clione. Two other feeding structures, chitinous hooks and the radula, extract the soft tissue from the prey’s shell to then be swallowed whole by the rhythmic movements of the radula and buccal mass muscles (Arshavsky et al., 1989).

Movement and Muscle Systems Generate Different Feeding Behaviors

The mouth of gastropods opens into the buccal cavity that is located within a muscular buccal mass (Figures 2A and 2B). An important structure within the buccal cavity is the radula, which is a flexible chitinous ribbon studded with rows of projecting teeth. The radula is attached to the odontophore (“tooth bearer”) where (depending on the species) it can rasp, bite or grasp the food, capture it, and pass it to the esophagus by a sequence of rhythmic movements. The odontophore is a complex mix of muscle and mechanical support structures. Some of these muscles are extrinsic (they originate in the body wall) and rotate the odontophore within the buccal cavity and others are intrinsic, some of which can move the radula relative to the rest of the odontophore. The organization of the muscles and their function in different species is most obvious in the muscles that control the movements of the radula. Their forms correlate with variation in the types of food they ingest. In contrast, the muscles that generate movements rotating the buccal mass complex forward and backward during the different phases of the ingestion feeding cycle are more similar in organization across gastropod species (apart from Navanax that has no radula or buccal mass).

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Figure 2. Biomechanical movements and muscle systems that generate different feeding behaviors. (A) Grazing in Lymnaea. (Ai) A three-phase feeding cycle (a fourth is a rest phase) underlies the grazing behavior of Lymnaea and other panpulmonate mollusks. During protraction (p) the odontophore and associated toothed radula rotate forward and protrude through the open mouth onto an algal substrate. Backward rotation during the first retraction phase (r1) rasps food into the buccal cavity. This is followed by a second swallow phase (r2) of backward rotation where the food is pushed into the esophagus. (Aii) The ventral base of the odontophore acts a fulcrum (upward arrowhead) about which it and the attached radula rotate forward and backward during the feeding cycle. The protractor posterior jugalis muscle (PJM) rotates the buccal mass forward during the p-phase, and the anterior jugalis rotates the buccal mass backward during the two retraction phases (r1-AJMlow; r2–AJMup). Three pairs of tensor muscles (Tensor) stretch the radula over the ondontophore during r1. (B) Grasping of seaweed by Aplysia. (Bi) A three-phase feeding cycle underlies rotational movements of the buccal mass in Aplysia. The radula of Aplysia has an opener and closure hingelike mechanism. The open radula is protracted through the open mouth to grasp the food (seaweed). The radula closes during r1 and is retracted into the buccal cavity. During r2 the food is hyper-retracted into the esophagus (Lu et al., 2015). (Bii) The radula is protracted through the mouth by the I2 muscle. Retraction of the radula/buccal mass is mediated by the I1/I3 muscle complex. The I5 muscle is an accessory radula closer muscle and the I4 a radula closer muscle. (C) Morphology of the buccal mass of Pleurobranchaea. Sequential stages of dorsal dissection shown from top to bottom of drawing. Muscle m1, ventral radula retractor; m2 lateral radula protractor; m3, dorsal radula protractor; m4, medial radula protractor; m5 buccal radula constrictor; m8, dorsal lip retractor (Croll & Davis, 1981). (D) Ingestion of prey in Navanax by pressure gradients. (Di) Pharyngeal muscle layers shown in cross-section of the pharynx. 1, serosal longitudinal; 2, luminal circumferential; 3, radial; 4, luminal circumferential; 5, luminal longitudinal. Dii) Movements involved in the capturing and swallowing of prey (Cappell et al., 1989).

Radula Rasping and Grasping

Rasping behaviors in panpulmonate freshwater snails such as Lymnaea and Helisoma (Rose & Benjamin, 1979; Murphy, 2001) begins when the animal opens its mouth and protracts the odontophore (p in Figure 2Ai). Simultaneously, the radula ribbon also protracts and is pulled over the tip of the odontophore. The radula teeth, arranged as a series of transverse rows along the length of the radula, are erected during this protraction process by their contact with the odontophore. During the r1 (rasp) phase the radula rotates backward, and its teeth rasp the food substrate so that algal particles are lifted into the buccal cavity (Figure 2Ai). Next the odontophore is further retracted during the r2 (swallow, hyper-retraction) phase, and the particles are pulled by the radula into the anterior region of the esophagus (Figure 2Ai). Thus, in panpulmonates the radula possesses the mechanical characteristics of both rasp (for excavation) and a conveyor belt for transporting particulate food to the esophagus. Cinephotographic recordings of Lymnaea (Rose & Benjamin, 1979) and Helisoma (Smith, 1988) shows that the radula slides over the underlying odontophore while the odontophore independently accelerates across the food substrate during each feeding stroke. This independent but concurrent movement of the radula and odontophore maximizes the food excavation from the food substrate, as demonstrated by Smith (1988).

Functional biomechanical studies of the feeding apparatus of Aplysia (Novakovic et al., 2006; Ye, Morton, & Chiel, 2006) clarified the control of the radula during grasping behavior. Aplysia feeds on stemlike seaweed, which it grasps with the radula (the “grasper”) and pulls it into the buccal cavity (Figure 2Bi). There is a pronounced groove in the dorsal surface of the odontophore that separates the left and right sides. This grove, together with the associated radula, opens and closes using a hingelike mechanism. During the protraction (p) phase of the feeding cycle (Figure 2Bi), the radula protrudes through the mouth in the open configuration. The lateral walls of the odontophore groove then close in apposition to each other, acting as a hinge, resulting in the grasping of the seaweed. The radula halves remain closed throughout the r1-phase that moves the food into the buccal cavity. A second retraction phase (r2) results in the ingestion of food into the esophagus (Figure 2Bi). There is no opener and closer radula-hinge mechanism in Lymnaea and other panpulmonates indicating an important evolutionary innovation in the biomechanics and muscle control in Aplysia.

The organization of muscles underlying grazing is comprehensively analyzed in Lymnaea (Carriker, 1946; Goldschmeding & de Vlieger, 1975; Rose & Benjamin, 1979). The odontophore is a large oval-shaped structure seen in side view (Figure 2Ai) with its ventral base acting as a fulcrum (arrowed in Figure 2Aii), about which it is rotated forward and backward during the feeding cycle by two sets of muscles that move the buccal mass in opposite directions (Figures 2Ai and 2Aii). The protractor posterior jugalis muscle (PJM) rotates the buccal mass forward during the protraction (p) phase of the feeding cycle. The anterior jugalis muscles (AJM) insert at this fulcrum point on each side and rotate the buccal mass backward during the two retraction phases of the feeding cycle, r1 rasp-phase (AJMlow), r2 swallow-phase (AJMup). Also inserted in the fulcrum are three pairs of tensor muscles (Tensor, Figure 2Aii), that wrap around the posterior surface of the odontophore to be inserted into the subradula membrane. Contraction of these muscles stretches the radula over the odontophore during r1, necessary for scraping the algal substrate during grazing.

The radula in Aplysia is protracted through the mouth by the I2 muscle (Figure 2Bii) that functions like the PJM of Lymnaea (Figure 2Aii). The radula is opened during protraction by I7, a muscle located deep within the odontophore, which is unique to Aplysia. A radula closer muscle, I4, lies beneath I2, and is a retractor muscle homologous to one of the radula tensor muscles, the supra-lateral radula tensor (SLRT) in Lymnaea (Crossley, Staras, & Kemenes, 2018) and Helisoma (Wentzell et al., 2009). The I5 muscle assists in radula closing. The radula also rotates about the hinge, which consists of the inter-digitation of the muscle fibers of several major muscles intrinsic to the radula. This muscle is absent in panpulmonates. Retraction of the radula/buccal mass in Aplysia is mediated by the I1/I3 complex of muscles, which lie in the same posterior area of the buccal mass as the AJM of Lymnaea and Helisoma (Figures 2Aii and 2Bii) and have the same function in withdrawing the food into the oral cavity by rotating the odontophore backward.

Pleurobranchaea grasps its prey by the cyclical eversion of a proboscis and the protraction of the radula through the open jaws (Figure 1F). Repeated protraction and retraction movements of the buccal mass pull the prey into the buccal cavity. Grasping of the food is carried out by a radula opening and closing mechanism similar to that of Aplysia (Croll & Davis, 1981). Radula protraction is due to contraction of muscles m2 and m4 that insert posteriorly on the radula sac (Figure 2C). Direct electrical stimulation of the m1 and m3 muscles result in retraction of the radula indicating that they play the role of radula retractors (ventral and dorsal, respectively). Muscle m5 is the most conspicuous muscle in the buccal mass, comprising a flattened sheet of tissue that encircles the anterior two thirds of the buccal mass (Figure 2C). It is a muscular constrictor that plays a role in grasping the food. The m5 muscle creases the radula and causes the radula teeth to hook the prey, gripping it tightly during the simultaneous pulling of the food into the gut by the radula retractor muscles. The m5 muscle in Pleurobranchaea functions to secure the prey that is extremely variable in size and liable to changes in shape due to compression. A muscle with this function is not present in Aplysia and other gastropods emphasizing the influence of the physical nature of the food type in the evolution of gastropod feeding.

Ingestion of Prey by Suction

Navanax carries out ingestion and swallowing by a system of muscular control that is entirely different in principle from the rest of the gastropods (Woollacott, 1974; Cappell et al., 1989). Other gastropods have numerous intertwined buccal mass muscles that interact to generate rotational movements of the odontophore and the rasping or grasping hingelike movements of the radula. In Navanax, the muscles involved in food ingestion consist of three mutually orthogonal muscles groups—radial, circumferential, and longitudinal—arranged in well-defined layers that are organized into layered bands around the lumen of the pharynx (Figure 2Di). These create pressure gradients that suck the prey into the pharynx and pass the food along the gut by peristaltic movements. There is no radula or odontophore. The different muscle types have different functions acting to expand the lumen of the pharynx (radial): to close the lumen (circumferential) of the pharynx or shorten and increase the diameter of the gut (longitudinal). Figure 2Dii illustrates the movements involved in capturing and swallowing prey. The pharyngeal lips protract toward the prey, and the rostral expansion of the pharynx seals the lips around the prey and sucks the prey into the pharyngeal cavity. The seal maximizes the negative pressure generated by contraction of radial muscles surrounding the pharynx. These along with the longitudinal muscle enlarge the lumen of the pharynx to create the general suction pressure for ingestion. Then the pharyngeal lips retract within the body cavity followed by a sequence of peristaltic movements that pass the food from the pharyngeal cavity through to the esophagus and then to more distal regions of the gut. Pharyngeal peristalsis involves the sequential circumference constriction, accompanied by a more caudal local expansion produced by radial muscle contraction, followed by more posterior constrictions in a repeating pattern of anterior to posterior contractions. Active expansion intensifies the pressure gradient facilitating the advance of the luminal content through the gut.

Relationships of Motoneurons to Muscles Correspond to Behavior

The motoneurons controlling the muscles of the buccal mass and other muscles involved in feeding movements are identified by examining the effects of motoneuronal spiking on the electrical responses of single muscle fibers (Figures 3Ai and 3Aii). The roles of the motoneurons in feeding were determined by recording their phase-dependent activity during food-induced feeding rhythms and showing by intracellular dye injection that they have the appropriate peripheral axonal projection along nerves that innervate the target muscle.

In Lymnaea, motoneurons active in all three phases of the feeding cycle are identified (examples in Figure 3Aiv) and their locations and axonal projections to buccal ganglion nerves determined by dye injection (Figure 3Aiii). The muscle recordings show that spikes in the motoneurons elicit 1:1 excitatory junction potential (EJPs) that summate to give an overall depolarizing wave (Figures 3Ai and 3Aii) that underlies muscular contraction. There is no evidence (Rose & Benjamin, 1979; Staras, Kemenes, & Benjamin, 1998) that the ejps trigger active membrane responses to evoke muscle action potentials.

Recordings in Aplysia from buccal mass protraction and retraction muscles and radula opening and closer muscles show similar non-spiking ejp responses (Church, Whim, & Lloyd, 1993). The different types of motoneurons have the appropriate anatomical nerve projections (Figure 3Bi) and firing patterns (Figure 3Bii) to be consistent with their functional roles as motoneurons for muscles active in the p, r1, and r2-phases of the ingestion cycle.

In Clione, motoneuronal control of hook and radula protraction and retraction movements are described (Figure 3Ci) (Norekian & Malyshev, 2006). The hook and radula movements occur in an alternating protraction and rasp pattern during prey acquisition (Figure 3Cii), and different hook and radula motoneurons control these movements (Figure 3Ciii). The function of the motoneurons was determined by stimulating the motoneurons using intracellular current injection and observing which of the muscles contracted. The hook motoneurons of Clione are absent in the Panpulmonata and other Euopisthobranchia and have evolved independently along with the appendages they control.

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Figure 3. Motoneuronal control of muscle contractions. (A) Lymnaea. (Ai) Intracellular muscle recording from a single muscle fiber in the PJM. Spikes in the B7a protraction phase motoneuron elicit 1:1 excitatory junction potentials (ejps). (Aii) r2-phase motoneuron B8 elicits 1:1 ejps in the AJMup (extracellular, top; intracellular bottom) (Aiii) Summary of the results from intracellular dye-injection experiments showing the peripheral nerve projections of buccal motoneurons. (Aiv) Summary of the motoneuronal innervation pattern of protraction, rasp (retract. 1) and swallow (retract. 2) phase muscles that underlie grazing in Lymnaea during a single feeding cycle. (B) Aplysia. (Bi) Motoneuronal axonal projections to muscles that are compatible with their roles in mediating muscular responses (Bii) Activity patterns of motoneurons during a single feeding motor program. (C) Clione. (Ci) Peripheral nerve axonal projections of radula protractor (RP) radula retractor (RR), hook retractor (HR) and hook protractor (HP) motoneurons. (Cii) Alternating protraction (p) and retraction (r) movements of the hook and radula that draw the prey into buccal cavity. (Ciii) Firing patterns of radula/hook protraction and retraction motoneurons during a single motor program. Note that hook and radula movements occur in an alternating protraction/rasp pattern during prey acquisition. Abbreviations: (Aiii) CBC, cerebro-buccal connective; DBN, dorso-buccal nerve; LBN, latero-buccal nerve; VBN, ventro-buccal nerve. (Bi) EN, esophageal nerve; RN, radula nerve. (Ci) hn, hook nerve; pn, proboscis nerve; rn1, radula nerve 1; rn2, radula nerve 2; sgn, salivary gland nerve.

Central Pattern Generator Circuits (CPGs) Produce Diverse Motor Patterns

Rhythmic feeding movements in gastropod mollusks result from neural-network oscillators that are located in the paired buccal ganglia (Figure 1B). They control rhythmic movements of the buccal mass and radula as well as associated digestion structures such as the esophagus and the salivary glands. The evolution of these circuits can account for the variety of motor programs required in gastropod species with different feeding behaviors. Organized oscillatory activity occurs in the absence of peripheral sensory input indicating the involvement of a CPG. As in other invertebrate systems (Katz & Hooper, 2007), rhythm generation in gastropods depends on a mixture of endogenous neuronal properties and network synaptic connections. The main differences in the organization of gastropod CPGs arise from variations in the flexibility of the feeding motor programs. The CPGs of the panpulmonates have an “automatic” program of activity consistent with their stereotyped ingestion behaviors whereas the CPGs of other gastropods (e.g., Aplysia have more “flexible” CPG programs consistent with their variable ingestion behaviors).

The Panpulmonate CPG

The rhythmicity of the feeding motor program in Lymnaea is generated by three types of interneurons, N1M, N2v, and N3t that are the core components of a tri-phasic oscillator (Figures 4Ai and 4Aii). The N1M, N2v, and N3ts fire in a three-phase sequence that is aligned to the protraction (p), rasp (r1), and swallow (r2) phases of the feeding cycle, respectively (Figure 4Aiii). The most important cells are the N1M and N2vs (Brierley, Staras, & Benjamin, 1997a; Brierley, Yeoman, & Benjamin, 1997c; Kemenes & Elliott, 1994) that occur in bilaterally symmetrical pairs in the left and right buccal ganglia (Figure 4Aii). Their firing alternates during the protraction (N1M) and rasp (N2v) phase of the feeding cycle (Figure 4Aiii). The rhythmic pattern of activity shown by N1M and N2v depends on their intrinsic plateauing properties (Figure 4Av), which provides the main oscillatory drive to the CPG network. The recurrent inhibitory synaptic connections between the two cells generate the sequence of N1M→N2v firing. The N1Ms continue to show plateauing properties in cell culture (Straub et al., 2002), and so the plateauing is truly endogenous whereas the N2vs require the presence of a chemical modulator, acetyl choline (ACh), and are thus “conditional” plateauing neurons. A three-neuron network is necessary to get the full three-phase CPG rhythm required for normal feeding behavior. This larger network includes the swallow phase CPG interneurons and the paired N3ts (N3tonics). N3t is not an endogenous oscillator but fires due to post-inhibitory rebound (PIR, Figure 4Av) (Elliott & Benjamin, 1985a) after receiving inhibitory synaptic input from the N2vs (Figure 4Aiii). By providing strong inhibition to the N1Ms during the swallow phase of the feeding rhythm, the N3ts delay the recovery of the N1Ms thus creating a separate swallow phase of the feeding cycle (Figure 4Aiii). No inhibitory synaptic feedback is present to stop the N2v firing and it is due to an endogenous hyperpolarizing mechanism. Biophysically accurate computational simulations of the Lymnaea feeding CPG successfully mimicked the main features of the system (Vavoulis et al., 2007), further validating the three-cell CPG network (Figure 4Aiv).

Motoneurons in Lymnaea play an important role in rhythm generation due to their electrotonic coupling with CPG interneurons (Staras et al., 1998). A significant feature of the coupling is that it is restricted to motoneurons and CPG interneurons that are active in the same phase of the feeding pattern. The B7a protraction phase motoneurons are coupled to the N1Ms, the B10 rasp phase motoneurons to the N2vs and the B4 swallow phase motoneurons to the N3ts (Staras et al., 1998). This coupling contributes to the same phase synchronicity in the feeding network but also makes it a component of the CPG; the motoneurons oscillate in a phase-locked manner with the patterned output to provide functionally relevant synaptic inputs to the CPG interneurons and reset phases of the feeding pattern. Finally, the endogenous properties of the motoneurons in Lymnaea are also important in rhythm generation. Swallow-phase motoneurons such as the B4/B8s are capable of bursting in culture in the absence of any CPG synaptic inputs, a further mechanism contributing to rhythmicity in the feeding network. This bursting is induced by the release of 5-HT from the CGC modulatory interneurons and is defined as “conditional” (Straub & Benjamin, 2001).

The feeding CPG of another panpulmonate, Helisoma, has a similar “automatic” tri-phasic pattern to Lymnaea when activated by food (Murphy, 2001). The interneurons that form the three subunits of the CPG in Helisoma (S1, protraction phase; S2, retraction 1; S3, retraction 2) fire sequences of bursts in each feeding cycle. How they contribute to the mechanism of rhythm generation is known in the B2 as a S2 phase interneuron, which has plateauing properties similar to those of the N2v cell of Lymnaea (Brierley et al., 1997a). B2 and N2v are both glutamatergic neurons (Quinlan & Murphy, 1991; Brierley et al., 1997b) providing further evidence that they are homologous. There are several types of S1 protraction-phase CPG interneurons, represented by the BCN1 interneurons, whose depolarization drives feeding motor programs. Their locations and axonal projections suggest that they are homologous with the N1Ms of Lymnaea. However, none of these BCN1 interneurons show the endogenous plateauing property of the N1Ms that is necessary for rhythm generation in Lymnaea (Kemenes & Elliott, 1994). The reciprocal inhibitory and excitatory synaptic connections between N1M and N2v, important for getting alternation of firing in the feeding CPG in Lymnaea, are also present in Helisoma (Figure 4B), confirming its general importance in determining the pattern of firing in the panpulmonate feeding CPG. There is no equivalent to the Lymnaea N3t swallow-phase CPG interneuron in Helisoma. The S3 phase neuron, N3a (Quinlan & Murphy, 1996), plays this role. PIR is present in this neuron but is neither sufficient nor necessary to get the S3 phase of spike activity (Quinlan & Murphy, 1996).

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Figure 4. Central pattern generator circuits. (A) Core CPG circuit of the grazing panpulmonate Lymnaea (Ai) Summary of the inhibitory (dots) and excitatory (bars) synaptic connections between the three types of interneuron, N1M, N2v and N3t that form the oscillator. (Aii) Axonal projections of the CPG interneurons. The N1M axon projects to the contralateral CBC whereas N2v has a peripheral axonal branch in the PBN. The N3t is a pure buccal interneuron. (Aiii) Simultaneous recording of the three CPG interneurons in one p→r1→r2 cycle. Synaptic connections are labelled on the recording. The N1M depolarizes N2v and triggers a delayed burst of spikes in N2v. (Aiv) Computational simulation of the Lymnaea CPG reproducing the same three-phase rhythm (modified from Vavoulis et al., 2007). (Av) Depolarizing pulses trigger plateaus in N1M and N2v. Release from hyperpolarization triggers PIR in N3t (modified from Brierley et al., 1997a; Straub et al., 2002; and Elliott & Benjamin, 1985b). (B) Core CPG network of the grazing gastropod, Helisoma. (C) Core CPG of Aplysia that generates a flexible grasping behavior. (Ci) B31/B32 and B63 interneurons operate as a protraction unit to alternate in a firing pattern with r1 B64 interneuron to generate a two-phase rhythm that underlies the initial grasp/bite behavior. B64 inhibits B31/32 and B63. B51 is active when a variety of sensory stimuli trigger the r2-phase to ingest food. Cii) Axonal morphology of the three CPG interneurons. B63’s axon projects to the contralateral CBC whereas B51 and B31/32 have a peripheral axonal branch. B64 is a pure buccal interneuron. (Ciii) Plateauing properties of the B31, B64 and B51 CPG interneurons (modified from Saada et al., 2009; Dickinson et al., 2015).

The Aplysia CPG

The analysis of the CPG in Aplysia has focused on the two-phase bite protraction (p)-swallow (r1) rhythm. The third phase (r2) is only required for a second swallow phase and is not an automatic part of the CPG motor program. The triggering of the third phase allows food that has entered the buccal mass cavity to move to the anterior gut (Cropper, Jing, & Weiss, 2017). Despite these differences between the CPG in Aplysia and the automatic three-phase CPG oscillator of the Panpulmonata there are individual neurons in the Aplysia CPG that have similar properties to those of Lymnaea and Helisoma.

Like the panpulmonates, plateauing of the Aplysia CPG interneurons (Figure 4Ciii) is an important part of the rhythm-generating mechanism with synaptic connectivity being the main determinant of the sequence of firing in the different phases of the ingestion cycle. Protraction is achieved when a plateaulike depolarization is generated in the B31/B32 neurons. They have a dual function acting as both interneurons and motoneurons. B63 is electrotonically coupled to B31/32 (Figure 4Ci) and provides part of the excitatory input and promotes synchronous firing between the two types of protraction-phase interneurons. In addition, there is chemical transmission between B63 and B31/32 (Figure 4Ci). B63 is a cholinergic interneuron, homologous to N1M, which induces both slow and fast excitatory EPSPs in B31/32. The slow muscarinic input induces inward currents in B31/32 that are necessary for plateau induction showing that plateauing is “conditional.” An interesting feature of the B31/32 is that they have positive feedback via muscarinic “auto” receptors (Saada et al., 2009). This plays an important role in triggering B31/32 plateaus and the activation of the protraction phase of the feeding cycle. Protraction is terminated by inhibitory feedback from a retraction phase CPG interneuron, B64 (Figure 4Ci) (Cropper et al., 2017) resembling N2v of Lymnaea (Figure 4Ai) and B2 of Helisoma (Figure 4B). However, the mechanism that produces the plateauing-induced burst of spikes in B64 is different. In Lymnaea it is due to an excitatory synaptic connection from N1M during protraction that triggers a delayed plateau on N2v (Figures 4Aiii and 4Aiv). In Aplysia an inhibitory synaptic input occurs on B64 during protraction and this is followed by a delayed burst of spikes. This is due to the reciprocal inhibitory connections between B63 and B64 (Fig. 4Ci) unlike the recurrent-inhibitory connections between N1M and N2v in Lymnaea (Fig. 4Ai). Reciprocal inhibition in CPG circuits is a well-established oscillatory mechanism generating alternating bursting patterns in invertebrate and vertebrate CPGs (Marder & Calabrese, 1996).

The r2-phase of the Aplysia feeding CPG cycle is recruited by artificial electrical stimulation of another plateauing neuron, B51 (Figure 4Ciii), a dual function interneuron/sensory neuron. B51 is active when the radula closer muscle (I4) contracts to signal the movement of food into the buccal cavity. This is a significant part of the mechanism for the triggering the enhanced hyper-retraction movements of the radula required for the passage of food into the esophagus. In isolated preparations, hyper-retraction is common when motor patterns are activated by stimulating the command-like neuron, CBI-4. CBI-4 stimulation induces B51 plateauing in the r2-phase of the feeding cycle (Jing et al., 2004; Sasaki et al., 2013) providing further evidence for the role of B51 in hyper-retraction. B51 connects to B64 by electrical and chemically mediated synapses (Fig. 4Ci) forming part of the three-phase CPG rhythm.

The Clione CPG

The feeding CPG of Clione consists of rhythmic protractor and retractor interneurons that control the ingestion movements of the radula (Arshavsky et al., 1989). The Bc-PIN (buccal protraction interneuron) cell body is located on the lateral edges of the buccal ganglion and its single axon projects to the opposite buccal ganglion and into the contra-lateral cerebro-buccal connective (CBC). In the protraction phase of the feeding cycle there is a depolarization followed by the triggering of a plateau in Bc-PIN followed by a hyperpolarization in the retraction phase that terminates firing. The retraction phase interneurons also show periodic bursting activity evoked by artificially depolarizing a neuron with a constant current. There is reciprocal synaptic inhibition between the protraction and retraction phase interneurons (Arshavsky et al., 1989) that have the dual function of generating the alternate pattern of firing and the triggering of firing in both cell types by PIR.

There are some features of Clione that indicate homologies with the CPG interneurons from other gastropods, which may be surprising given the striking differences in behavior in the two species. However, differences in the endogenous properties of the Clione retraction-phase interneurons (no plateauing like the retraction neurons of Lymnaea, Helisoma, and Aplysia) and synaptic circuitry (reciprocal rather than recurrent inhibition) suggest evolutionary divergence.

The Initiation of Feeding Motor Programs by Higher-Order Interneurons: Comparison of Selection Mechanisms

Higher-order interneurons that drive CPG activity and respond to sensory cues necessary for feeding behavior are good candidates for a role in the initiation of rhythmic motor behaviors (Kupfermann & Weiss, 1978). Cerebro-buccal interneurons (CBIs) with these properties are present in gastropods with a variety of feeding behaviors. CBI cell bodies are located in bilateral symmetrical groups in similar locations in the left and right cerebral ganglia (Figure 5Ai, Figure Bi, Figure Ci, Figure Di, and Figure Ei). Their numbers vary between 13 and 18 in different gastropod species (Aplysia, Lymnaea, Limax, Pleurobranchaea, and Clione). They have a characteristic anatomy with single ipsilateral axon that projects to the buccal ganglion on the same side. The initiating functions of the CBIs is best understood in Aplysia and Lymnaea. Focusing on these two species gives an insight into the evolution of commandlike initiating mechanisms in different types of feeding behaviors, one more flexible than the other.

In Lymnaea, maintained depolarization of the CV1a (Figure 5Aii) or the electronically coupled CA1 (Figure 5Aiv) consistently drives a strong three-phase rhythm in the CPG network (Figure 5Aii) as does the CT2 CBI interneuron (Benjamin, 2012). The related CV1bs are also capable of driving a feeding rhythm in a previously non-rhythmic preparation or increasing the frequency of an already existing rhythm but they are much weaker in their effect. They tend to fire in a continuous manner (McCrohan & Kyriakides, 1989) unlike the CV1as that fire rhythmically during the protraction phase of the feeding rhythm (Figure 5Aiii) (McCrohan, 1984). The CV1as drive the CPG because of a strong monosynaptic excitatory synaptic connection with the N1M (McCrohan, 1984). They are phase-locked to the feeding pattern due to inhibitory feedback (Figure 5Aii) from CPG interneurons active during the N2 phase of the feeding rhythm. The CBWC is also able to drive a feeding rhythm in a quiescent preparation (McCrohan & Croll, 1997) but has inhibitory effects on already- active preparations causing a reduction in the frequency and intensity of the feeding rhythm. These dual actions are reproduced by the application of the CBWC neuropeptide APGW to an isolated CNS preparation. Simultaneous recordings of Lymnaea CBIs show that they are co-activated during a sucrose-driven rhythm (Figure 5Aiii). Their commandlike effects are combined to drive the feeding motor program. Suppressing activity in individual CBIs during the food activation of feeding does not block the feeding response (Benjamin, 2012).

In Aplysia, the CBI-2 is used to drive ingestion programs (Figure 5Bii) like the CV1a and CA1 neurons of Lymnaea. However, unlike Lymnaea the CBI-2 requires co-activation with other CBIs to drive strong ingestion rhythms. Ingestion is facilitated by co-activating CBI-3 (APGW peptide-containing interneuron) (Morgan et al., 2002). CBI-11 has a similar role to CBI-3 except that CBI-11 can weakly drive ingestion cycles on its own (Wu et al., 2014). This is different from the CV1a or CA1 cells of Lymnaea that drive a full and reliable ingestion-feeding program without a partner CBI. CBI-2 also interacts with CBI-12 to produce variants of the protraction phase of the motor pattern (Jing & Weiss, 2005) leading to greater flexibility of the feeding behavior. They are activated together by application of seaweed to the lips (Figure 5Biii). This is an example of the role CBIs play in the more flexible feeding behavior of Aplysia that is absent from the Lymnaea feeding system. CBI-4 is another example of an Aplysia CBI that is absent in Lymnaea. Its role is to stimulate the r2 (hyper-retraction) phase of the feeding rhythm (Jing et al., 2004; Sasaki et al., 2013), that is otherwise absent in the more typical two-phase feeding program of Aplysia.

The CBIs of Pleurobranchaea (Paracerebral Neurons) also initiate fictive feeding programs in isolated preparations (Kovac et al., 1982) and their size, morphology, and similar locations on the cerebral ganglia provide evidence for their homology with the commandlike CBIs of Aplysia and Lymnaea. Most attention has focused on neurons that are phasically active during these feeding rhythms, the paracerebral phasic neurons (the PCP) (Gillette et al., 1982). Tonically active CBIs (PCT) in Pleurobranchaea are less effective in driving feeding rhythms (Kovac et al., 1982), which resembles the situation in Lymnaea where the CV1b (tonic) CBIs have similar weaker triggering effects on the buccal motor program. There are subpopulations of PCp neurons (Kovac et al., 1983) unique to Pleurobranchaea that also have a commandlike capacity. These provide excitatory synaptic inputs to the phasic PCp neurons. The Polysynaptic Excitors (PSE) provide polysynaptic chemically mediated synaptic input to the PCp neurons (Figure 5Cii), and the two ETiis are electrotonically coupled to the same neurons (Figure 5Cii) and play a role in synchronizing the spiking activity in the PCp population.

Limax CBIs from a phasically active group (Figures 5Di and 5Dii) have different effects on feeding activity (Delaney & Gelperin, 1990). CB1 reliably triggers bouts of fictive feeding when stimulated individually or in pairs, like the CBIs in other gastropods. CBEC is not effective alone in triggering feeding, although it may influence feeding activation due its electrotonic coupling with CB1. Moderate frequency stimulation in CB4 speeds up bite rate, while the same stimulus applied to CB3 slows biting. The effects of stimulating the inhibitory CB3 neuron in Limax resembles the APGW-mediated inhibitory effect of the CBWC in Lymnaea.

One bilaterally symmetrical CBI (Cr-BM) has been identified in Clione (Figure 5Ei) that drives coordinated activation of the neural networks controlling the main feeding structures, the buccal cones, chitinous hooks, and the radula (Figure 1G). This CBI controls the complex feeding appendages of Clione. Mediated by excitatory and inhibitory synaptic connections to motoneurons (Figure 5Eii), activation of Cr-BM drives feeding responses that are part of the ingestion behavior, such as opening of the skin folds that cover the mouth, rhythmic muscular contractions of the buccal mass, and coordinated protraction–retraction movements of the hooks and radula (Norekian & Malyshev, 2005). Cr-BM is GABAergic and exogenous application of GABA mimics the effects of neuronal stimulation. The GABAergic CBI-11 of Aplysia produces excitatory effects on the buccal motor programs and its similar morphology, location on the cerebral ganglion and its physiological effect provide evidence that it is homologous with Cr-BM in Clione.

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Figure 5. Initiation of feeding motor programs by the cerebro-buccal interneurons (CBIs). (A) Lymnaea CBIs. (Ai) Location of CBIs in the left cerebral ganglion. Axons of the CBIs project into the CBC en route to the ipsilateral buccal ganglion. (Aii) Artificial depolarization of CV1a in the isolated CNS drives fictive feeding in the N1M, N2d and N3p CPG interneurons. CV1a bursting is phase locked to the protraction phase of the feeding pattern (modified from Kemenes et al., 2001). (Aiii) A food stimulus (sucrose) applied to the lips in a semi-intact preparation activates a feeding pattern in three co-recorded CBIs. Extracellular recording of the CBC shows simultaneous axonal bursting activity in a population of CBIs (modified from Benjamin, 2012). (Aiv) Summary of CBI excitatory connections with the buccal ganglia (dashed lines) N1M interneuron. Reciprocal electrical synaptic connections (resistor symbol) link the CBIs (CV1a, CA1, CBWC) that fire in the same protraction phase of the feeding cycle. Chemosensory inputs from lip sensory neurons (SN) converge on the CBIs, driving feeding rhythms. Red-circled CBIs are able to drive motor programs by triggering N1M plateaus. (B) Aplysia CBIs. (Bi) Locations of CBIs in the cerebral ganglion and their projections to the CBC. (Bii) Artificial activation of CBI-2 drives fictive feeding in buccal ganglion feeding interneurons/motoneurons. (Biii) Seaweed application to the mouth activates CBI-2 and CBI-11 for the duration of biting response (modified from Jing & Weiss, 2005). (Biv) Summary of CBI synaptic connections with protraction-phase interneurons and motoneurons in the buccal ganglia (dashed lines). Reciprocal electrical synapses connect CBI-2, CBI-3, and CBI-11. Sensory neurons providing excitatory stimulation of feeding converge on the CBIs. (C) Pleurobranchaea CBIs (Ci) Map of CBI locations and axonal projections. (Cii) The PCP and PCT CBIs are commandlike neurons. The PSE and ETii influence these commandlike functions via synaptic connections (chemical and electrical) with the PCP neurons. There are further types of influential non-CBI interneurons (I2, I1, and MSE) that have synaptic connections with the PCP. (D) Limax CBIs (Di) Map of CBIs, axons projecting to the CBC (Dii) CB1 fires phasically and acts as a commandlike CBI, synaptically exciting buccal neurons. CB4 speeds up bite rate in an already active preparation. Both CB4 and the inhibitory CB3 have biphasic inhibitory/excitatory synaptic connections with buccal neurons producing complex responses on feeding output. CBEC is electrically coupled to CB1 and influences feeding activation via this indirect route. All Limax CBIs receive convergent food-activated chemosensory input to influence feeding. (E) A single characterized Clione CBI, the Cr-BM (Ei) Cr-BM location and axonal CBC projection as well as other neurons labelled via backfilling the CBC. (Eii) Cr-BM has commandlike and organizational functions on the complex feeding motor programs of Clione and has direct excitatory and inhibitory synaptic connections with CPG interneurons (Bc-PIN) and motoneurons (radula, RR, RP; hook, HP, HR).

Modulation: Conserved Mechanisms Across Species

Modulatory interneurons provide dynamic control of the gastropod feeding network. They may be “extrinsic” or “intrinsic” to the feeding network. Extrinsic modulation causes global changes to influence the whole feeding circuit, whereas intrinsic modulation causes local changes in the feeding circuit.

Extrinsic Modulation: A Global Feeding Arousal System

The CGCs (Cerebral Giant Cells in Lymnaea; Metacerebral Cells in Aplysia; and Cerebral 1, C1s in other gastropods) were one of the first cell types classified as homologous in gastropod mollusks (Weiss & Kupfermann, 1976). They are a bilateral symmetrical pair of giant serotonergic neurons whose cell bodies lie in a characteristic location in the left and right cerebral ganglia (shown on the left side in Figure 5Ai, Figure Bi, Figure Ci, Figure Di, and Figure Ei). Their single large axons project ipsilaterally along the CBCs to the buccal ganglia where they have synaptic connections with all of the identified buccal ganglion feeding interneurons and motoneurons (Figure 6D) (McCrohan & Benjamin, 1980b; Yeoman, Brierley, & Benjamin, 1996).

The CGCs have a permissive (gating) role in the generation of feeding but cannot “command” activity in the CPG at physiological firing rates (Jing, Gillette, & Weiss, 2009). Tonic firing activity provides background excitatory modulation to the feeding network, which reduces the threshold for feeding—an example of an arousal response (Jing et al., 2009). In Lymnaea, by recording the CGCs in intact animals using fine-wire recording, the arousal effects of CGC on the feeding system are shown to depend on their tonic firing rates (Yeoman et al., 1994). During ingestive feeding behavior, the CGCs fire at ~20 spikes/minute (Figure 6Ai). And below this range of firing, for instance during locomotion (Figure 6Aii) and quiescence (Figures 6Aiii and 6Bi–iii), feeding responses to food are absent. In both Lymnaea and Aplysia, the CGCs also influence the frequency of the feeding rhythms above the threshold level of firing, a second type of modulation (Yeoman et al., 1994, 1996). Required for CGC modulation is 5-HT (serotonin, the CGC’s transmitter) (Yeoman et al., 1994). The 5-HT2 receptor antagonist cinanserin reversibly blocks feeding rhythms from the effects of CGC tonic on rhythm generation.

Two types of mechanism targeting CPG interneurons underlie the modulatory role of the CGCs in gating (Yeoman et al., 1996). One involves the increased level of “background” synaptic depolarization provided by the increase in the tonic firing rate of the CGCs in feeding snails (McCrohan & Benjamin, 1980b; Yeoman et al., 1994, 1996). The other depends on the effects of CGC firing on the endogenous properties of the CPG interneurons. The main CGC synaptic connections are with the N1M and N2v cells (Yeoman et al., 1996). Post-synaptic depolarization reduces the threshold for plateauing in both neuron types. The N1Ms are slowly depolarized by CGC stimulation (Yeoman et al., 1996), making them more likely to respond to other types of plateauing-triggering synaptic input (e.g., from the CBIs). Long-term effects of CGC tonic firing are most significant for the N2v’s role in gating. Suppressing CGC activity for two minutes leads to a complete loss of N2v plateauing, only recovering when CGC firing is allowed to recommence, demonstrating that CGC activity is necessary for the endogenous plateauing of N2v (Yeoman et al., 1996).

The post-synaptic effects of CGC tonic firing on motoneurons are also important in network gating (McCrohan & Benjamin, 1980b). The resulting synaptic depolarization reduces their spike threshold and makes them more responsive to CPG synaptic inputs that drive their rhythmic firing (Benjamin et al., 1981). Without this modulatory synaptic input the level of motoneuron firing is insufficient to drive the muscular activity required for feeding movements (Benjamin & Elliott, 1989). Gating of network activity is also due to the CGC modulatory effects on the endogenous properties of motoneurons. In culture and in the intact ganglion, evoking brief bursts of CGC spike activity or applying 5-HT to the neurons causes modulatory effects on the rasp and swallow phase motoneurons (B4/B8) that can last for minutes. The induction of endogenous bursting and an enhancement of PIR all increase the probability of B4/B8 firing during feeding cycles contributing significantly to the gating function of the CGCs (Straub & Benjamin, 2001).

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Figure 6. Extrinsic modulation by the serotonergic Cerebral Giant cells (CGCs). Examples from the Lymnaea feeding system (A) Fine wire recordings of CGC firing rates in vivo during different behaviors. (Ai) Extracellular recording during a bout of feeding on a lettuce leaf. CGC firing rates were greatest during this behavior (~15 spikes/min). (Aii) Spike rates were lower when the snail was locomoting around the tank but not biting (~7 spikes/min). (Aiii) During quiescence, the CGC only fired occasionally and no bites were recorded. (B) Rates of CGC firing were artificially altered by current injection to match those occurring in the intact animal. (Bi) At a CGC firing rate of 15 spikes/min, the SO was able to drive a high-frequency rhythm. (Bii) At the CGC threshold level, 7 spikes/min, a lower frequency feeding rhythm occurred showing frequency-dependence. (Biii) In the absence of CGC firing only weak feeding rhythms could be activated. (C) The role of 5-HT in CGC modulation. (Ci) In normal saline CGC firing supports high frequency SO driven fictive feeding. (Cii) The 5-HT antagonist, cinanserin, blocks feeding responses. (Ciii) The response recovers after washing in normal saline. (D) Summary of the CGC synaptic connections to motoneurons (B1, B2, B4, B5, B6, B7) and CPG interneurons (N1M, N2d, N2v, N3p, N3t) and the SO. Bar represents excitatory connection, black dot an inhibitory connection. One of the connections is biphasic (N3p) (modified from Benjamin, 2012). (Ei) In Lymnaea, suppressing SO spiking activity during a food-induced rhythm results in a slow, irregular rhythm. (Eii) Plotting the cycle period with and without SO activity demonstrates the SO’s role in maintaining the frequency and regularity of the CPG driven feeding pattern (modified from Kemenes et al., 2001).

Intrinsic Modulation: Control of Network Frequency and Stability

In a number of invertebrate motor systems, modulatory interneurons that drive CPG activity receive feedback from the CPG interneurons they control, resulting in the entrainment of their spike activity to the motor rhythm. This type of reciprocal interaction provides positive feedback within the feeding network and plays a role in maintaining rhythmic feeding patterns (Gillette et al., 1978). An example of such a mechanism is the Lymnaea SO interneuron. This single asymmetric cell, located in either the left or right buccal ganglion, initiates feeding patterns in the CPG when artificially stimulated into tonic activity by current injection (Figure 6Bi). Once the feeding rhythm commences in the CPG, the SO becomes rhythmically active during the protraction phase of the feeding cycle due to inhibitory feedback from CPG interneurons such as N2v and N3t (Yeoman et al., 1993). The SO has no endogenous capability to oscillate (Straub et al., 2002). The SO has strong excitatory monosynaptic connections with N1M and in SO-driven rhythms fires just before it in the same protraction phase of the feeding rhythm (Elliott & Benjamin, 1985a). The SO thus provides another component of the depolarizing synaptic connection (along with CV1a) that triggers the N1M plateau. The lack of SO activity in a sucrose-driven rhythms or the suppression of firing by the application of hyperpolarizing current does not prevent the occurrence of a feeding pattern in the CPG (Figure 6Ei) indicating that the SO is not part of the CPG and its firing unnecessary for feeding initiation (Kemenes et al., 2001). However, in the absence of SO spiking, rhythmic activity in the CPG slows in frequency and becomes irregular (Figures 6Ei and 6Eii). Stimulation of the SO by current injection to fire at different rates shows that both frequency control and maintenance of the regular feeding pattern depend on the SO (Rose & Benjamin, 1981). The data indicate that the core CPG oscillator alone is not able to generate the regular high-frequency rhythm observed by strong feeding stimuli such as sucrose in the intact animal. The function of the SO to maintain and control the frequency of the feeding cycle by reducing the duration of the protraction phase is supported by computational modelling of the synaptic connections and firing patterns of the SO and CPG interneurons (Vavoulis et al., 2007).

An evident feature of the B50 modulatory buccal interneuron of Aplysia (Dembrow et al., 2003), is its similarity to the SO of Lymnaea. B50 shares a number of characteristics with the SO providing evidence for homology. They are both unpaired neurons possessing “loop” morphologies. When stimulated they both elicit rhythmic motor programs in the buccal ganglia, without activating CBI-2 (Dembrow et al., 2003) or its Lymnaea homologue CV1a (McCrohan, 1984), by exciting protraction phase CPG interneurons (B31/32 and B63 of Aplysia) with hexamethonian-sensitive EPSPs (excitatory post-synaptic potentials) (Yeoman et al., 1993). They also both receive similar excitatory synaptic inputs from their CGC homologues. There is one difference between the B50 and SO. B50 is excited by CBI-2 (Dembrow et al., 2003) but the SO received no synaptic input from its Lymnaea homologue, the CV1a neuron (McCrohan, 1984).

Hunger and Satiety: Evolution of Responses to Food Stimuli

Hunger and satiety are motivational/behavioral states that contribute to the flexibility of feeding behavior in gastropods. These behavioral states strongly influence the animal’s responses to food in the consummatory phase of feeding irrespective of the type of feeding behavior. In grazing panpulmonates such as Lymnaea, the “spontaneous” feeding movements that form part of their foraging behavior are enhanced in food-deprived snails compared with satiated snails. Most information on the evolution of neural circuit mechanisms underlying hunger and satiety states originate from two gastropod species, Lymnaea and Aplysia, allowing species with different feeding behaviors to be compared.

Appetitive Behavior of Lymnaea

Hunger level influences the frequency of spontaneous rasping movements, appetitive feeding cycles, which occur in the absence of food and are greater in food-deprived compared with well-fed snails. This depends on the level of motivation state (Tuersley, 1989). For example, snails deprived of food for four days, showed a higher number of spontaneous rasps than those deprived for one day (Crossley et al., 2016). This motivational effect persists in isolated ganglia preparations and an in vitro correlate can be recorded as changes in spontaneous fictive feeding activity in motoneurons. Preparations from food-deprived animals produce more spontaneous fictive feeding cycles than their fed counterparts (Tuersley, 1989). Given that these neural correlates of hunger and satiety are present in the completely isolated ganglion preparation, the motivational effects must be due to retained central mechanisms that control spontaneous feeding activity. One such central controller of spontaneous feeding is the N3t CPG interneuron. The N3ts fire tonically to inhibit the N1M CPG cell and the rate of this tonic activity affects the frequency of the feeding rhythm in the whole feeding CPG (Staras et al., 2003). The N3t firing rate is higher in ganglia from satiated (Figure 7Ai) compared with snails that were food-deprived for one day (Figure 7Aii). After four days of food deprivation the number of appetitive bites increased further. However, tonic activity was not reduced compared with one-day food-deprived animals. Instead N3t’s PIR activity was reduced, increasing the likelihood of a subsequent appetitive cycle being generated (Figures 7Aiii and 7Aiv) (Staras et al., 2003; Crossley et al., 2016). Thus, changes in the level of inhibition on the system serve as a state-dependent mechanism to control food-searching behavior.

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Figure 7. Hunger and Satiety influences feeding behavior. (A) Lymnaea’s appetitive feeding behavior. (Ai) In satiated snails, N3t’s high tonic firing suppresses activity in the N1M CPG interneuron (N3t→N1M inhibition, thick red line) preventing cycles. (Aii) In one-day food-deprived snails, N3t tonic inhibition is lower (thin red line) allowing occasional spontaneous cycles. Post-cyclic N3t PIR prevents a second cycle being generated (thick red line post cycle). (Aiii) In four-day food-deprived snails, reduced N3t PIR allows a higher level of cyclical firing in N1M (modified from Crossley et al., 2016). (Aiv) Schematic of the effects of hunger-state on spontaneous appetitive feeding cycles, and the associated relationships with the levels of N3t’s tonic and PIR activity. (B) A satiety mechanism in Lymnaea based on gut expansion (Bi) Semi-intact preparation (left) used for testing the effects of esophageal stretching on the firing of the esophageal mechanoreceptors (OM) mimicking the effects of gut expansion caused by bulk food ingestion (right). (Bii) Intracellular dye injection reveals the anatomy of the OM. It has bilateral projections to the DBNs and innervates the pro-esophagus. (Biii) Stretching the pro-esophagus causes phasic somatic spikes in the OM. (Biv) Evoking spikes in the OM inhibits the SO and blocks feeding patterns in the B4 motoneuron (modified from Elliott & Benjamin, 1989). (C) Neural network mechanisms underlying hunger and satiety in Aplysia. (Ci) In satiated animals, the inhibition of food intake is due to the activation of B20 interneurons. ApNPY neuropeptide activates B20 via both the esophageal nerve and B18. The excitatory synaptic response that CBI-2 has on B40 in the presence of food is blocked by the strong inhibitory effects of apNPY. (Cii) In hungry Aplysia, B40 is activated by the CBI-2 synaptic pathway, promoting food intake. B20 is inactive under these food-deprived circumstances. At the start of a meal, when the animal is hungry, the CBI-2→B40→Food intake pathway acts to drive food ingestion. As the meal proceeds the B18/apNPY→B20 satiety pathway gradually takes over to prevent food intake (Jing et al., 2007).

Consummatory Behavior in Lymnaea and Aplysia

Satiety in the consummatory phase of feeding in Lymnaea is mediated by mechanosensory responses to food ingestion in the anterior part of the gut (Figure 7Bi). In Lymnaea, three to five esophageal mechanoreceptors (OMs) occur in each buccal ganglion and their axons project to and innervate the pro-esophageal region of the gut that lies immediately behind the buccal mass (Figure 7Bii). The OMs respond to gut expansions with brief bursts of high-frequency firing (Figure 7Biii) (Elliott & Benjamin, 1989). They adapt their firing rates during artificially maintained steady distensions (Figure 7Biii), but during the more rhythmic gut movements observed during the peristaltic movement of food through the anterior gut they maintain their responsiveness. The OMs in Lymnaea have extensive inhibitory connections with many neurons of the feeding circuit (examples in 7Biv) including CPG interneurons (Elliott & Benjamin, 1989). Evoking repeated bursts of spikes in the OMs at frequencies similar to those recorded during gut expansion inhibit a SO-driven rhythm indicating that the OM mechano-sensory responses to bulk food stimuli are sufficient to mediate satiety in the intact animal (Figure 7Biv).

In Aplysia, a large meal produces a state of satiation characterized by a lack of locomotion and a failure to elicit biting responses to food stimuli (Kupfermann, 1974). Further work shows that this is due to the presence of bulk food in the anterior gut (Susswein & Kupfermann, 1975). Injection of high-viscosity non-nutritive bulk, which filled the anterior gut, is capable of causing satiation but similar injections in the posterior gut did not, showing that satiety is due to activation of mechanoreceptors associated with expansion of the anterior gut, rather than chemical stimulation. It is probable that there are mechanoreceptors in the gut of Aplysia similar to those found in Lymnaea, although they are yet to be identified by direct recording. As in Lymnaea, the sensory signals from homologous peripheral mechanoreceptors travel to the buccal ganglia via axons in the esophageal nerves (Kuslansky, Weiss, & Kupfermann, 1987). Cutting the esophageal nerves results in a significant increase in the volume of food needed to satiate the animals, but eventually they cease feeding to prevent bursting of the gut.

In vertebrates and invertebrates, several “satiety” peptides have been implicated in the inhibitory processes leading to the development of satiety states. The injection of the Aplysia homologue of NPY, Aplysia neuropeptide Y (apNPY), reduces food intake and slows down the rate of ingestion (Jing et al., 2007). apNPY is localized in the buccal ganglion afferents originating in the esophageal nerve that is known to be involved in hunger and satiety. During the early stage of a meal, food expansion of the esophagus activates mechanosensory fibers in the esophageal nerve leading to a progressive increase in the release of apNPY into the feeding circuit. In the presence of apNPY, CBI-2 stimulation reduces the fraction of ingestion responses, therefore reducing food ingestion, and increases the fraction of intermediate/egestion responses. apNPY released from gut afferents inhibits the ingestion-driving neuron B40, while simultaneously activating the egestion neuron, B20 (Figure 7Ci). Separate gut afferents activate B18, which releases apNPY and drives further activity of B20. This provides evidence for a satiety mechanism that involves changes in the configuration of the feeding CPG itself, resulting in the eventual termination of food intake. Although a NPY-like peptide (LyNPY) is present in Lymnaea, injection does not inhibit food intake (de Jong-Brink, ter Maat, & Tensen, 2001). The lack of a role of LyNPY in satiety is consistent with the absence of the Aplysia B18 and B40-like interneurons in Lymnaea.

Similar to other feeding systems, glucose levels in the hemolymph of Lymnaea vary according to feeding state. Glucose concentrations change dramatically from 15µg/ml in starved animals to 760µg/ml in those that are well fed (Scheerboom, Hemmings, & Doderer, 1978). Consistent with a role in satiety in Lymnaea, application of D-glucose at concentrations similar to those found in satiated snails, hyperpolarized a number of isolated feeding neurons sufficient to stop them firing. Significant among those showing this inhibitory response were the CGC and SO modulatory interneurons (Alania, Dyakonova, & Sakharov, 2004). Spiking activity in both of these cell types is necessary for feeding responses to food. It is important to note that glucose does not influence feeding behavior in Aplysia and plays no role in satiety (Horn, Koester, & Kupfermann, 1998). Thus, glucose injected into the hemocoel of Aplysia has no effect on meal size, bite latencies, swallowing rate, or food intake.

Hunger in Lymnaea promotes feeding due to a reduction of the tonic inhibitory modulation of the CPG by N3t (Staras et al., 2003). Reducing inhibition of the CPG allows the CV1a commandlike CBI to initiate ingestion feeding rhythms. The reduction of spontaneous inhibitory synaptic inputs to the CV1s in hungry snails also plays a role (Whelan & McCrohan, 1993). In Aplysia, hunger promotes food intake by a synaptic pathway mediated by the food-activation of CBI-2 that excites B40 and inhibits B20 (Figure 7Cii).

Changes in the Perception of Food Stimuli

In Lymnaea, satiated animals are more likely to reject a stimulus that their food-deprived counterparts will attempt to ingest, suggesting that their perception of the stimulus changes based on their hunger state (Crossley et al., 2018). This decision generalizes to stimuli of different sensory modalities. Sensory re-tuning does not underlie the animal’s altered perception since hunger state does not affect the output of sensory neurons involved in the detection of the stimuli. Instead, central reconfiguration of the feeding network biases the animal from egestion toward ingestion cycles as the level of food deprivation increases. Egestion driving interneurons (PRNs, pattern reversing neurons, Figure 9Di–iv) encode the animal’s hunger state and are in a relative upstate in satiated animals, biasing cycles toward egestion. By blocking the output of these neurons in vivo, satiated animals are more likely to ingest a stimulus that they previously deemed inedible. This central switch therefore provides the animal with a simple mechanism by which it can lower its threshold for promoting ingestion behavior when the animal is hungry, increasing the likelihood of successful foraging behavior. These findings resemble those found in Aplysia (Jing et al., 2007) where, as a meal progresses and the animal reaches satiation due to gut distention, more egestion cycles are generated. However, in Lymnaea the bias toward egestion in satiated animals persists in the absence of any sensory stimulation from the gut and is present in animals food deprived for two days, past the point at which the gut would be distended by food. Therefore, Lymnaea utilizes a combination of central and sensory processes to modify its appetitive behavior based on its hunger state: N3t and PRN act centrally to modify aspects of the animals’ foraging behavior in order to maximize food localization, and OM signals gut distension, terminating feeding due to satiation.

Multitasking: Comparisons of Behavioral Switching

Many motor systems are multifunctional meaning that they are capable of performing more than one behavioral task. Using motor program-switching mechanisms animals can achieve a greater behavioral repertoire within the constraints imposed by a fixed endowment of neurons and muscles. Thus, different motor programs are selected according to the current behavioral needs of the animal. In gastropod mollusks alternate feeding behaviors, classified as either ingestion or egestion, are generated by sensory switching of the central motor programs. Ingestion is elicited when appetitive stimuli (e.g., food) are applied to the lips or tentacles whereas the presence of an aversive stimulus applied to the lips or to the inside of the gut (e.g., the imbibing of an object too large to be swallowed or a toxic chemical stimulus) elicits egestion. All the gastropods of neurobiological interest exhibit both behaviors (Euopisthobranchia: Aplysia, Morton & Chiel, 1993a, 1993b; Pleurobranchaea, Croll & Davis, 1981, 1982. Panpulmonata: Helisoma, Murphy, 2001; Ramakrishnan, Arnett, & Murphy, 2014; Lymnaea, Crossley et al., 2018). The mechanisms underlying the species-specific switching of ingestion to egestion reverse the direction of the movement of food through the mouth from inward to outward result from differences in the sequencing and timing of radula movements. Species-specific circuit and muscular mechanisms involving motoneurons, interneurons and their synaptic connectivity cause the motor programs of radula movements to allow different types of behavioral switching to occur.


In Aplysia, the longitudinal fold in the center of the radula acts as a hinge and allows the radula to open and close. The timing of these opening and closing movements determines whether the feeding behavior is ingestive or egestive. During ingestion behavior the radula protracts open and retracts closed to grasp the food (Figure 8Ai) whereas during egestion the radula protracts closed and retracts open so that the food is pushed out of the mouth (Figure 8Aii). Underlying these differences in behavior are the opener (I7) and closer (I4) radula muscles that switch their activity to generate the two mutually exclusive behaviors. Other types of buccal mass muscles determine the position of the radula during the protraction/retraction cycle and as the same pattern of contractions is required for both behaviors, they do not change their phase of activity when the behavior switches between ingestion and egestion.

Because the timing of the opening and closing of the radula is the main difference between egestion and ingestion, this aspect of neuronal control has been the focus of attention. One of the key motoneurons B8, which mediates radula closing, fires during both ingestion and egestion (Figures 8Bi and 8Bii). Other motoneurons are behavior-specific such as radula opener motoneurons B48 and B44. B48 mediates radula opening during ingestion but not during egestion whereas B44 mediates radula opening during egestion but not during ingestion (Friedman et al., 2009). The differences in the two types of patterning results from different mechanisms. Changes in excitability mediate behavioral specificity in B48. The excitability of B48 decreases during egestion (Friedman & Weiss, 2010) whereas it increases during ingestion (Friedman et al., 2015). By contrast, the changes in the firing of B8 are mediated by behavior-specific interneurons such as B4/B5 and B20 (Jing & Weiss, 2001). For example, B20 and B4/5 both promote egestive activity in B8 (Figure 8Bii). B20 increases B8 spike activity during protraction and B4/5 decreases it during retraction. The CBIs also play an important role in multitasking. Recruitment of CBI-3 by CBI-2 electrotonic coupling promotes B8 activity in the retraction phase of ingestion by activating the B40 interneuron (Jing & Weiss, 2002) (Figure 8Bi).

More research on multi-tasking is required understand how the Aplysia generates another type of feeding behaviors such as grazing on algal substrates observed under field conditions (Kupfermann & Carew, 1974).

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Figure 8. Multitasking: switching from ingestion to egestion in food “grasping” gastropods (A) Aplysia. (Ai) During ingestion the radula protracts open through the open mouth and is then retracted closed, pulling the food in. (Aii) During egestion, the radula is closed during protraction and opened during retraction, pushing the food out of the mouth (still images from Neuroscience Online video. (Bi) Interneuronal control of radula closer motoneuron B8’s firing during ingestion. Recruitment of CBI-3 by CBI-2 electrical coupling excites B40 and inhibits the egestion network, promoting retraction phase firing in B8. (Bii) During egestion, B20 drives B8 activity during protraction, and B4/5 inhibits it during retraction. Orange circles/lines signify neurons/connections preferentially active in each behavior; gray signifies neurons/connections not active. (C) Pleurobranchaea. (Ci, Cii) Summary of muscular control of ingestion and egestion in Pleurobranchaea. The black bars represent the relative strengths (height) and duration (width) of activity during the two behaviors. Radula protractor (m2, m4), retractor (m1, m3), and constrictor (m5) muscles are all active during ingestion. During egestion, protraction-phase activity is increased whereas retraction-phase activity is reduced, and m5 constrictor activity is almost completely absent. The dorsal lip retractor muscle (m8) remains the same in both behaviors.


Both ingestion and egestion movements in Pleurobranchaea, entail repeated protraction and retraction movements of the radula (Croll & Davis, 1981, 1982). During active ingestion of squid pieces, the food object is positioned in the center of the fold as part of the grasping mechanism and the radula is opened and closed by mechanisms that are similar to those of Aplysia. However, as part of the grasping mechanism the buccal mass radula constrictor muscle creases the radula and causes the radula teeth to hook the prey, gripping it more tightly during the simultaneous pulling of the food into the gut by the retractor muscle (m3). The evolution of this constrictor function also extends to the egestion retraction movements of the radula (Croll & Davis, 1982). Instead of constriction due to activity in m5 (Figure 8Ci), the radula remains broad and flattened, because m5 is silent during egestion (Figure 8Cii). In addition, the radula does not role inward during egestion because of the substantially reduced activity in the m3 retractor muscle. The flattening and the lack of in-folding of the radula as well as the closure of the jaw near the beginning of retraction ensure that food objects are pushed out of the mouth during protraction. In contrast to ingestion, the power stroke of egestion is the protraction phase as seen by weaker retractor muscle activity (e.g., m3) compared with the more intense, longer discharges in the radula protractor muscles (e.g., m4) (Figures 8Ci and 8Cii).


During ingestion in Lymnaea, the radula is protracted from and then retracted back into the mouth moving anteriorly toward the dorsal mandible (Figure 9Ai, left). This effectively scoops food into the mouth where it is retracted further toward the esophagus. In the presence of an aversive stimulus Lymnaea performs egestion behavior. During egestion the radula is protracted out of the mouth from an anterior region, moving in a posterior direction away from the dorsal mandible where it is then retracted back into the mouth (Figure 9Aii, right). Significantly, during protraction, the dorsal edge of the radula is drawn across the roof of the dorsal food channel, expelling any objects from within the buccal cavity out of the mouth.

Similar differences in radula movements occur between the ingestion and egestion behaviors in Helisoma (Figures 9Bi and 9Bii) (Arnett & Murphy, 1994). Watermelon extract acts as a potent ingestive stimulus (Figure 9Bi). Applying an emetic, Listerine, to the mouth immediately triggers observable reversed movements of the odontophore, characteristic of egestion (Figure 9Bii). The power stroke of the odontophore switches from retraction during ingestion to protraction during egestion. Thus, during egestion, the protraction phase is greater in duration. Also, the positions of the fulcrum, the point around which the odontophore rotates, during the protraction/retraction cycle reverse in direction to allow regurgitation through the mouth rather than swallowing into the esophagus. One striking difference between the two behaviors in Helisoma (Murphy, 2001) is the absence of the hyper-retraction (r2) phase during egestion (Figure 9Bii), which is always present during ingestion (Figure 9Bi). Thus, switching the feeding cycle from a three- to a two-phase rhythm. Modification of the feeding cycle makes functional sense because it prevents the toxic chemicals transferring to other parts of the animal.

The neural basis for the absence of the r2-phase during egestion is investigated in Helisoma by carrying out recordings from identified feeding neurons while applying Listerine to the esophagus of a semi-intact preparation (Figure 9Biii) (Ramakrishnan et al., 2014). The strong protraction cycles that occur during egestion behavior are correlated with prolonged action potential bursts in the protraction phase interneuron type, the N1a, which is known to drive spike activity in protraction phase motoneurons. Listerine perfusion shuts down the r2-phase excitatory CPG-driven inputs to the r2-phase B19 motoneuron so that they become inactive during this phase of the feeding cycle while the S2 inhibitory (r1) synaptic inputs are retained (Figure 9Biii), thus showing the selective effects of toxic stimuli on behavioral selection. It is possible that the inhibition of the r2-phase of feeding could occur in other panpulmonates but is difficult to observe because of the opaqueness of the buccal mass. In Aplysia, the inhibitory interneuron B70 could be playing a role in suppressing hyper-retraction. It is active during egestion and has a “late” inhibitory effect on the firing of the B8 closer motoneuron that coincides with the r2-phase of the feeding cycle (Sasaki et al., 2009). A neuron with a similar role is unknown in other gastropods.

Ingestion and egestion in panpulmonates share some common elements with each other. For instance, in both behaviors the sequence of radula protraction/retraction remains the same. However, by co-recording from sets of muscles in a reduced preparation in Lymnaea, it is shown that SLRT activity varies between ingestion and egestion (Figures 9Ci and 9Cii) (Crossley et al., 2018). During ingestion, SLRT activity coincides with retraction phase muscle, AJM. However, during egestion, SLRT activity precedes that of the AJM, occurring during the protraction phase. Contraction of the SLRT during the protraction phase causes a dorsal movement of the odontophore—contributing to the radula being in apposition to the dorsal food channel of the buccal cavity and exiting the mouth from the anterior region to play a role in food rejection. A SLRT motoneuron, B11, identified in the buccal ganglia of Lymnaea (Crossley et al., 2018) is homologous with B8 of Aplysia. Similar to B8, B11 is active during both ingestion and egestion cycles (Figures 9Ciii and 9Civ) and its phase of activity switches between behaviors, matching the activity patterns observed on the SLRT muscle itself. Notably, the core feeding CPG is active during both ingestion and egestion and does not alter its phases of activity, thus driving the basic protraction/retraction sequence. Higher-order elements of the feeding network are differentially recruited into each behavior and shape the final outcome. CV1a, which drives robust ingestion cycles, is inhibited by synaptic inputs during egestion cycles (Figure 9Civ). The dopaminergic PRNs (Figure 9Di) in the buccal ganglia were identified as members of the egestion-driving network in Lymnaea (Crossley et al., 2018). They are active during sensory driven egestion (Figure 9Dii), and activation of a single PRN is sufficient to drive egestion cycles (Figure 9Diii). The PRNs are homologous to the egestion driving neurons B20 in Aplysia. Both neurons have a similar location and morphology, and both utilize dopamine as a transmitter. Notably, activation of PRN causes large waves of inhibitory inputs on the ingestion-driving neuron, CV1a (Figure 9Div). These inputs are polysynaptic in nature but are also present during sensory-driven egestion. Therefore, elements of the egestion network actively inhibit those of the ingestion-driving network, providing a simple mechanism for behavioral selection between incompatible behaviors (Figure 9E).

Gastropod Feeding Systems: Evolution of Neural Circuits that Generate Diverse Behaviors

Figure 9. Multitasking: switching from ingestion to egestion in “grazing” gastropods. (A) Lymnaea. (Ai) During ingestion the radula is protracted through the open mouth and then retracted back into the mouth moving anteriorly toward the dorsal mandible. (Aii) During egestion the radula protracts out of the mouth from an anterior region, away from the dorsal mandible, and then retracted into the open mouth (modified from Crossley et al., 2018). (B) The cycle of odontophore and radula movements in Helisoma observed in transparent newly hatched snails (Bi) Ingestion stimulated by watermelon extracts. Forward protraction (p) movements are followed by backward retraction (r1) and swallowing (r2) movements. (Bii) Listerine triggers egestion. The p-phase is greater in duration than during ingestion, and the r2-phase is absent, switching the feeding cycle from a three- to a two-phase rhythm. (Biii) Listerine application blocks the r2-phase excitatory input to the B19 motoneuron (black arrow) leading to inactivity. The r1 inhibitory input is retained. Thus the r2 CPG component necessary for swallowing behavior is blocked (modified from Ramakrishnan et al., 2014). (C) Neural and muscular control of ingestion and egestion in Lymnaea (Ci) During ingestion, SLRT activity coincides with the retraction (r) phase activity in AJM. (Cii) During egestion, SLRT activity occurs in the protraction (p) phase, preceding the AJM. (Ciii, Civ) B11 is a SLRT motoneuron and its activity corresponds with activity in the SLRT in the two types of behavior. (D) Lymnaea PRNs. (Di) Immunostaining of a dye-marked PRN shows the presence of dopamine (DA). (Dii) PRN is active during sensory-driven egestion. (Diii) Artificial activation of PRN is sufficient to drive egestion cycles (modified from Crossley et al., 2018). (Div) Activation of PRN inhibits the ingestion driving CBI, CV1a, via a polysynaptic pathway. (E) Model of the interactions between egestion and ingestion networks. Elements of the egestion pathway inhibit the ingestion-driving network providing a mechanism for behavioral choice between incompatible behaviors.

Conclusions and Challenges

The aim of this article is to relate neural circuit evolution to behavioral evolution in gastropod feeding systems. It is conceptually reasonable to explore how the evolution of behavior involved changes in neural circuitry to alter behavior (Katz, 2016). The results on the feeding systems of gastropod mollusks provide evidence for this concept because the evolution of different types of feeding behaviors in this diverse group of mollusks is mirrored by species-specific changes in the neural circuitry some as a result of re-purposing, others because of the development of new types of neurons. Homologous neurons are identified in different gastropod species across the behavioral spectrum, and this facilitates the comparison of neural circuits and their evolution across species.

Behavioral Evolution

The clearest example of evolution of feeding behaviors is in the variations in the role of the radula (to rasp, bite, or grasp the food), the most important organ involved in food consumption. Radula movements have evolved in accordance with the type of food consumed. Species-specific external appendages (e.g., the proboscis and tentacle-like buccal cones) also evolved to allow the capture of specialized food items, particularly in carnivorous gastropods. Another major factor in behavioral evolution is the flexibility of feeding behaviors. Gastropods that feed on food items (plants and animal prey) that vary in size, shape, and compressibility have more flexible behaviors than those species that feed on uniform food substrates.

Table 1. Summary of Homologies in Gastropod Feeding Systems













AJM low

AJM low



AJM up

AJM up



























































Homology of Muscular Control

The muscles that control the stereotyped feeding behavior in the panpulmonate snails, Lymnaea and Helisoma, have the same anatomy, biomechanics and movement patterns to be classed as homologous. These are the muscles (PJM, AJMlow, AJMup, Table 1) that control the protraction, rasp (r1) and swallow (r2) phases of the feeding cycle, respectively. The radula tensor (SLRT) muscle is also homologous in the two species (Table 1).

Aplysia has a different, more flexible behavior than the panpulmonates. It has muscles that open (I7) and close (I4) the radula and allow the grasping of their stemlike seaweed food. The I4 is homologous to the SLRT muscle in Lymnaea and Helisoma (Table 1) but has a different function. In contrast, the I7 is not homologous with any of the muscles in Lymnaea and Helisoma. Other muscles that have functions in rotating the odontophore forward and backward in all three species (protraction phase, PJM/I2; retraction phase, AJM/I1/I3) are all homologous with their panpulmonate cousins with similar functions in moving the radula/odontophore complex. The nudipleuran gastropod, Pleurobranchaea, also uses opener and closer muscles to grasp food but developed a new type of radula constrictor muscle that is absent in other gastropods.

It can be concluded that the homologous muscles in gastropods with the same behaviors have the same functions. This is also the case for gastropods with different behaviors when the movements are the same across species. However, when a muscle generates an alternative type of behavior, the function of the homologous muscle is different, which is an example of the evolution of muscular control that is coincident with behavioral evolution. There are examples in one of the Euopithobranchia where none of the muscles have been shown to be homologous with other gastropods. The lack of muscular homology is an example of the major changes in muscular organization that generate the completely different and unique behaviors of carnivorous gastropod mollusks such as Navanax.

Homology of Neurons and Synaptic Connections

Homology is investigated at the single identified neuron level in all of the functional elements of the gastropod feeding network. Again, the question arises concerning the functions of homologous neurons in gastropod species with a wide range of feeding behavior. Neurons are classified as homologous based on shared characteristics such as soma size, location, neurotransmitter content, anatomy, electrophysiological properties such as endogenous firing, and synaptic connectivity.

Protraction and retraction (r1 and r2) phase motoneurons have similar cell body locations, firing patterns, and axonal projections in gastropods with the same (e.g., Lymnaea and Helisoma) and different behaviors (e.g., Aplysia), and they have the same functions. These homologous motoneurons innervate homologous muscles that generate movements in the same phase of the feeding cycle (Table 1). However, one type of homologous motoneuron, the B11 of Lymnaea and the B8 of Aplysia, that innervate the SLRT and I4 muscles, respectively, fire in different phases. They fire during either the protraction phase or the retraction phase depending on whether the feeding behavior is egestive or ingestive, respectively. Thus, they play an additional role in multitasking (Table 1). The homologous B7a and B31/32 motoneurons in Lymnaea and Aplysia, respectively, are members of the CPG circuit and are also multifunctional.

The functional relationships of homologous neurons in the CPG rhythm-generating circuits are more complex and depend on whether the feeding behavior is stereotyped (e.g., panpulmonates) or more flexible (e.g., euopisthobranchs). The r2-phase of the feeding cycle is often absent from flexible feeding programs (e.g., Aplysia), and the CPG interneurons that control this phase of ingestion behavior are variably programmed into the feeding rhythm depending on whether the food item is palatable or not. Homology is most obvious within the groups of protraction phase and r1-phase interneurons of the CPG (Table 1). Homologous CPG interneurons of both types share within-group axonal morphologies and the intrinsic electrical ability to generate plateau potentials. This is the case of gastropods with the same (Lymnaea and Helisoma) and different (Aplysia, Clione) feeding behaviors. Also, the chemical synaptic transmitters of homologous CPG interneurons are the same in different types of behavior: ACh is the transmitter for the protractor interneurons (N1M of Lymnaea; B63 of Aplysia) and glutamate for the r1-phase interneurons (N2v of Lymnaea; B2 of Helisoma; B64 of Aplysia). However, the synaptic connections between the protraction and r1 components of CPG networks show differences. The panpulmonates, Lymnaea and Helisoma both have recurrent inhibitory connections as part of their rhythm-generating mechanism. However, Aplysia and Clione have reciprocal inhibitory connections between protraction and r1 interneurons—a different type of rhythm generation mechanism. More information is required from a wider range of gastropods to understand why these differences in connectivity have evolved in these different species. There are no homologies between the hyper-retraction CPG interneurons. They appear to have evolved independently in the three species that have been investigated. The main conclusion that emerges from this discussion of CPG homology is in the variability of the intra-circuit synaptic connectivity. This applies to gastropods with both similar and different behaviors. For the CPG interneurons that generate the r2-phase of the feeding cycle, the re-purposing of synaptic connectivity generates a more flexible type of feeding behavior.

Homologous CBIs of the type whose function is to initiate ingestion motor programs are found in gastropods with both similar (Lymnaea, Limax) and different (Aplysia, Pleurobranchaea, Clione) behaviors (Table 1). These commandlike CBIs (CBI-2; CV1a; PCp) all drive species-specific feeding motor programs. A second group of homologous CBIs (CBI-11; CA1; Cr-BM) (Table 1) also drive feeding to complement the effects of the first group; however, when activated alone their effects are usually weaker than those of the first group (details of this are unknown for Clione). A third type of homologous CBI (CBWC; CBI-3; CB3) (Table 1), has mixed inhibitory/excitatory effects on the ingestion rhythm depending on the frequency of firing. The CBWC of Lymnaea drives a feeding rhythm at low frequency but switches to inhibition at high frequency. Inhibition is the predominant effect on an ongoing feeding rhythm of the homologous CB3 of Limax. Aplysia’s CBI-3 induces a feeding rhythm when co-activated with CBI-2 but when stimulated alone inhibits ingestion. In the flexible feeding motor program of Aplysia, combinations of activity in different homologous interneurons are required to initiate ingestion-like programs. Stimulation of CBI-2 only results in a two-phase p and r1 motor program. Another CBI type, CBI-4, functions to drive the third r2-phase of ingestion. Further flexibility arises when CBI-2 is co-stimulated with CBI-12. This leads to a different type of motor program. The CBI types, CBI-4 and CBI-12, which play such an important role in flexibility in Aplysia, are absent in the Panpulmonata, whose “automatic” three-phase motor program always follows CV1a/CA1 activation.

Extrinsic modulation by the homologous CGC/MCCs occurs in all gastropods examined irrespective of whether they have the same or different feeding behaviors (Table 1). They function as an arousal system to facilitate the response of the feeding network to food stimulation. Their retained location, axonal anatomy, electrical properties, and network functions in all these species suggest they are not subject to the evolutionary selection processes that led to changes in neural circuitry observed in other parts of the feeding network. Homologous intrinsic modulatory neurons are present in panpulmonates and euopisthobranchs. The SO of Lymnaea and B50 of Aplysia share a number of characteristics providing detailed evidence for homology.

Two homologous interneurons, B20 in Aplysia and the PRN in Lymnaea, play a key role in hunger and satiety and multitasking. Their status as homologues is justified by their similar cell body locations and axonal morphology. In addition, they are both dopaminergic and have identical electrical synaptic connections with homologous CPG interneurons: B63 in Aplysia and N1M in Lymnaea. PRN and B20 are both important for encoding hunger state and in satiated animals suppress food ingestion. They are both excited by esophageal sensory pathways that drive egestion. So, as well as mediating satiety in the feeding circuit they also switch feeding behavior from ingestion to egestion.

Gastropod Neuronal Homology and Gene Profiling: Future Directions

The investigation of similar gene expression patterns helps to recognize homologous cell types in the nervous system (Striedter et al., 2014). In gastropods, the ability to progress this type of molecular analysis to single identified neurons is vital in improving the knowledge of neuronal homology (Moroz et al., 2006). Information based on gene profiling improves the probabilistic argument for homology based on other anatomical, pharmacological, and electrophysiological methods. By sequencing cDNA libraries from the CNS, it has been possible to identify a large number of expressed sequence tags (ESTs) encoding mRNAs from several gastropod species (Aplysia, Moroz et al., 2006; Lymnaea, Sadamoto et al., 2012; Biomphalaria, Adema et al., 2017). Single neuron profiling of identified Aplysia neurons has taken this process a stage further (Moroz et al., 2006). Motor and sensory neurons from the gill-withdrawal circuit revealed cell-specific mRNA enrichment for mRNAs encoding neuropeptides and other molecules involved in cell–cell signaling. Analyzing cDNA libraries from single MCC serotonergic neurons, obtained examples of both common and rare species-specific transcripts. The subcellular transcriptome of the MCCs also contains mRNAs for several cell communication components such as protein kinase C and the NMDA receptor that are known to be important in signaling in gastropod mollusks (Marra et al., 2013; Brierley et al., 1997b). The presence of neuron-specific rare transcripts is important because these transcripts act as a “signature” for the classification of homologous neurons in different species. According to Moroz (2018), the availability of high-throughput genomics will allow the capture of virtually all the neurons from gastropods such as Aplysia. This opens remarkable possibilities for future studies of homologies and their evolution if this methodology is applied to the other model gastropod systems with categorically related behaviors such as swimming or feeding. So far, there have not been any studies on gene profiling in comparative homology. But this approach is necessary for a deeper understanding of the relationship between neural network evolution and behavioral evolution.


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