COMMENTARY Two pairs of tentacles and a pair of …The Pulmonata is a subclass of the class...

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IntroductionThe Pulmonata is a subclass of the class Gastropoda in the phylumMollusca. It contains several orders, such as Stylommatophora(Limax, Achatina, Helix, etc.), Basommatophora (Lymnaea,Helisoma, etc.) and others (Klussmann-Kolb et al., 2008). We focushere on the Stylommatophora, a group of terrestrial pulmonates,i.e. land slugs or snails, possessing slender eye-bearing stalks.Terrestrial pulmonates have two pairs of tentacles: superior andinferior pairs. Their eyes are at the tips of the superior pair, but bothpairs are equipped with sensory epithelia. In some studies, thesuperior and inferior tentacles are also called the posterior andanterior tentacles, respectively. Although the tentacles subserve thedetection and transduction of visual, olfactory and mechanosensorystimuli to the central nervous system, terrestrial pulmonates seemto employ olfaction as their primary means of perceiving theexternal world (Chase, 2002).

Terrestrial pulmonates acquire mammalian-quality learning withodors as conditioned stimuli (CSs) and consolidate what they havelearned into long-term memory (Gelperin, 1975; Matsuo et al.,2002). For example, in Limax, olfactory-aversion learning occurswhen an odorant CS is paired with an aversive unconditionedstimulus (US) (Gelperin, 1975; Sahley et al., 1981). A natural foododor, such as that of carrot or cucumber, is used as the CS whereasquinidine sulfate, carbon dioxide or electrical shock is used asthe US. A long-term memory is formed after a single pairedpresentation of a CS and a US, and it is retained for more thanseveral weeks (Gelperin, 1975; Matsuo et al., 2002; Matsuo et al.,2010a).

To elucidate the cellular and molecular mechanisms underlyinglearning and memory in terrestrial pulmonates, researchers have sofar focused on the functions of the tentacles and procerebra (e.g.Gelperin, 1999; Watanabe et al., 2008; Matsuo and Ito, 2011).

However, we still face the question of why terrestrial pulmonatesinevitably possess two pairs of tentacles and a pair of procerebra.In a certain sense, such structures appear futile for the life ofpulmonates. Here we exemplify and discuss both the functionaloptimization and structural redundancy of two pairs of tentacles anda pair of procerebra involved in olfactory learning by terrestrialpulmonates, in particular Limax (Fig.1). Before starting ourcommentary, we acknowledge that much of the work of RonaldChase serves as a solid foundation for the present commentary.

Two pairs of tentaclesThe tentacle as a versatile sensory organ

Pulmonates use their chemical senses to find food and to avoiddanger, regardless of whether they live on land or in water(Gelperin, 1974). Several locomotive patterns are elicited bychemical senses, e.g. food finding, homing and escape. Chemotaxisis the phenomenon in which pulmonates direct their movements inconcentration gradients of certain chemicals, such as odors orpheromones, in their environment. Chemotaxis is called positive ifthe movement is in the direction of a higher concentration of thechemical in question, such as a chemical in food, and negative ifthe movement is in the direction of a weakening concentration, suchas a source of danger.

Pulmonates use their tentacles primarily to determine theorientation to distant food sources (Croll, 1983) (Figs1 and 2).Experiments in which chemical stimulants are applied locally to thetentacles and in which receptors in the tentacles are histologicallycounted showed that tentacles have a special chemosensitivity(Croll, 1983; Emery, 1992; Chase and Tolloczko, 1993; Cummiset al., 2009). Lesion experiments on tentacles have alsodemonstrated this chemosensitivity: early research by Chase andCroll (Chase and Croll, 1981) showed that the removal of either or

The Journal of Experimental Biology 214, 879-886© 2011. Published by The Company of Biologists Ltddoi:10.1242/jeb.024562

COMMENTARY

Two pairs of tentacles and a pair of procerebra: optimized functions and redundantstructures in the sensory and central organs involved in olfactory learning of

terrestrial pulmonates

Ryota Matsuo, Suguru Kobayashi, Miki Yamagishi and Etsuro Ito*Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Shido, Sanuki 769-2193, Japan

*Author for correspondence ([email protected])

Accepted 7 December 2010

SummaryTerrestrial pulmonates can learn olfactory-aversion tasks and retain them in their long-term memory. To elucidate the cellularmechanisms underlying learning and memory, researchers have focused on both the peripheral and central components ofolfaction: two pairs of tentacles (the superior and inferior tentacles) and a pair of procerebra, respectively. Data from tentacle-amputation experiments showed that either pair of tentacles is sufficient for olfactory learning. Results of procerebrum lesionexperiments showed that the procerebra are necessary for olfactory learning but that either one of the two procerebra, rather thanboth, is used for each olfactory learning event. Together, these data suggest that there is a redundancy in the structures ofterrestrial pulmonates necessary for olfactory learning. In our commentary we exemplify and discuss functional optimization andstructural redundancy in the sensory and central organs involved in olfactory learning and memory in terrestrial pulmonates.

Key words: learning, olfaction, procerebrum, pulmonate, tentacle.

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both of the superior tentacles prevents the terrestrial snail Achatinafrom locating food.

The superior tentacles are extremely sensitive to stimulation bywinds. In the terrestrial snail Achatina, gentle air puffs to thesuperior tentacle evoke transient discharges in the tentacularganglion at both stimulus onset and stimulus offset (Chase, 1981).In Limax, a gentle wind (without odor) induces electro-olfactogramoscillations in the olfactory epithelium of the superior tentacle, andthis oscillation affects olfactory information processingdownstream (Ito et al., 2006). Therefore, the tentacles function notonly as olfactory organs but also as mechanosensors, and there areinteractions between these two sensory modalities, such as chemo-sensing and mechano-sensing. In fact, the presence of windenhances the accuracy of the chemotactic behavior of insects(Willis et al., 2008).

Eyes are present only in the superior tentacles, but their functionsseem to be used only for phototaxis. Limax use their eyes fornegative phototactic behavior (Crozier and Federighi, 1924;Matsuo et al., 2002; Yamagishi et al., 2008). Optic nerves sendprojections to the cerebral ganglion, although the nerve bundles arenarrower than the olfactory pathways in Helix (Ovchinnikov,1986). Taken together, these results indicate that the two pairs oftentacles of terrestrial pulmonates serve as versatile sensorystructures to detect multimodal environmental stimuli.

Roles of bilateral tentaclesBecause the tentacles are present as bilateral pairs, pulmonates canshow tropotaxis. This is the movement of a pulmonate towards oraway from a stimulus as the pulmonate compares sensory inputsfrom the paired receptors on both sides of the body. However, thetentacles can also function independently on each side. This enablesklinotaxis, a locomotive mode based on a sampling of externalstimuli successively in time during pulmonate movement.

Chase and Croll designed experiments using unilateral lesions ofthe superior tentacles to determine which of these strategiesAchatina uses (Chase and Croll, 1981). The results depended onenvironmental conditions. When the snails were required to orientthemselves in the absence of any wind, they needed the pair ofsuperior tentacles, implying the use of tropotaxis. Because thecomparison of olfactory information from the bilateral tentacles isa prerequisite for tropotaxis, these results indicate that the two sides

of the olfactory system exchange olfactory information, probablyvia the cerebral commissures. However, when orienting to anupwind odor source, a single superior tentacle is sufficient,indicating that the snails use klinotaxis in the presence of mechano-stimulation.

A new insight into klinotaxis was recently obtained fromDrosophila. In Drosophila larvae, a unilateral olfactory organ issufficient for chemotactic locomotion along an odor concentrationgradient, probably via klinotaxis. But bilateral olfactory inputsenable the larvae to navigate in more complex, challengingenvironments, such as in a shallow, linear spatial concentrationgradient of odorant molecules (Louis et al., 2008). Louis et al.proposed that bilateral olfactory inputs are integrated into the neuralcoding spikes with a higher signal-to-noise ratio (by a factor of √2)than the case of a unilateral input (Louis et al., 2008). Although itis currently unclear where the bilateral olfactory inputs areintegrated in the brain of the slug (or even in Drosophila larvae),a similar enhancement mechanism might work for odor detectionin terrestrial pulmonates.

As described earlier, terrestrial pulmonates only use their eyesfor phototaxis. Terrestrial pulmonates also use a tropotactic strategyto avoid light places. They seem to compare the intensities of visualinputs from the right and left eyes, and move towards the darkerside. If Limax has a unilateral eye removed, it rotates in thedirection of the removed eye (Crozier and Federighi, 1924).Similarly, in Helix, if its superior tentacle is anesthetizedunilaterally, it often rotates towards the direction of the amputatedside (Friedrich and Teyke, 1998). This behavior may be anotherexample of tropotaxis in pulmonates (i.e. negative phototaxis). Thepossession of bilateral tentacles is obviously a prerequisite for atropotactic strategy of locomotion, whether it is driven by olfactoryor visual information.

R. Matsuo and others

Fig.1. The terrestrial slug Limax valentianus. In conditioning experiments,we usually use adult slugs at ~12–16 weeks after hatching (0.5–1g). IT,inferior tentacle; ST, superior tentacle.

Fig.2. Semi-intact preparation including the superior and inferior tentaclesand the central nervous system of L. valentianus. IT, inferior tentacle; MLN,medial lip nerve; SE, sensory epithelium; ST, superior tentacle; TN,tentacular nerve. Scale bar, 1.0mm.


881Tentacles and procerebra in Pulmonata

Structural features of superior and inferior tentaclesThe sensory structure of a tentacle includes a sensory epithelium,a tentacular ganglion and a tentacular nerve connecting thetentacular ganglion to the cerebral ganglion (Fig.3). The sensoryepithelium is at the tip of the tentacle. The tentacular ganglion isbeneath the sensory epithelium, and it ramifies into digit-likeextensions over the sensory epithelium. Studies using silver-stainedsections of the tentacles or rhinophores of gastropods (Lane, 1962;Emery and Audesirk, 1978) revealed that bipolar sensory neuronsare distributed beneath the sensory epithelium. The majority ofthese bipolar neurons form synaptic connections (i.e. synapticglomeruli) with neurons whose cell bodies are located in the digitsof the tentacular ganglion, whereas only ~10% of the axons of thesebipolar neurons travel directly to the cerebral ganglia (Chase,2002).

Retrograde staining of tentacular nerves showed that manysensory neurons and interneurons in the digits of the superiortentacles have axons that terminate directly in the cerebral ganglion(Chase and Tolloczko, 1993). Most of the olfactory inputs fromsensory neurons are synaptically relayed at the tentacular ganglionand enter into the procerebrum whereas mechanosensory afferentsare thought to directly project into the metacerebrum, a distinct partof the cerebral ganglion (Rogers, 1971; Ierusalimsky and Balaban,2010). The procerebrum, therefore, serves as a third-order level ofolfactory information processing (see A pair of procerebra:Structure and function of procerebra).

Although there are no essential differences between therepertoires of the types of neurons in the superior and inferiortentacular ganglia (Chase and Kamil, 1983a; Chase and Kamil,1983b; Ito et al., 2000), Ito et al. found that there are four structuraldifferences between the two pairs of tentacles by means ofbackfilling of the tentacular nerves: (1) fewer fibers form the thickfiber tracts in neuropils in the superior tentacles than in the inferiortentacles; (2) there are more stained neurons in the superiortentacles than in the inferior tentacles; (3) the ratio of stainedneurons to total neurons is lower in the superior tentacles than inthe inferior tentacles; and (4) as with the digits, the tentacularganglion is partially covered by a single cell layer in the inferiortentacles whereas the superior tentacles lack such a cell layer.

The anatomical projection patterns to the cerebral ganglion areroughly identical between the superior and inferior tentacles (Chaseand Tolloczko, 1993). However, Ierusalimsky and Balaban(Ierusalimsky and Balaban, 2010) recently demonstrated that thereare also some differences in the way superior and inferior tentaclesproject into the cerebral ganglion of Helix. The entire neuropil area(the terminal mass layer) of the procerebrum receives projectionsfrom the tentacular nerves (superior tentacle) whereas only thedistal one-third of it receives projections from the medial lip nerves(inferior tentacle). These anatomical differences might explain thefunctional and physiological differences between the superior andinferior tentacles described below.

Functional differences between the pairs of tentaclesThus far, it has been shown that the superior pair is useful forsensing airborne chemical signals and for directing the pulmonate’slocomotion whereas the inferior tentacles are particularly importantfor following mucus trails and other chemical cues that lie on thesubstrate. If Achatina has either one or both of its superior tentaclesamputated and the air is still, it cannot locate food placed distantly(Chase and Croll, 1981). Thus, the superior tentacles are necessaryfor tropotaxis. However, the mucus tracing behavior is abolishedby inferior tentacle amputation but not by superior tentacle

amputation (Chase and Croll, 1981). This suggests that the twopairs of tentacles function differently in the behavior of terrestrialpulmonates, although both serve as chemosensory organs.

Friedrich and Teyke demonstrated in Helix that superiortentacles are necessary and sufficient for locating food to which thesnails have been appetitively conditioned whereas the inferiortentacles, but not the superior tentacles, are necessary and sufficientfor the acquisition of food-attraction learning (Friedrich and Teyke,1998). This result is another example of the functional differencebetween the two pairs of tentacles. Although both pairs canpotentially detect the odor molecules derived from food duringconditioning and memory retrieval, and can potentially access theprocerebrum, where memory formation is thought to take place, theresults of Friedrich and Teyke (Friedrich and Teyke, 1998) indicatethat each pair is specialized to the distinct pulmonate behaviorrelated to learning and memory.

Physiological differences between two pairs of tentaclesFor the most part, the procerebrum receives olfactory inputsindirectly via the tentacular ganglion, and the oscillatory frequencyof the local field potential (LFP) in the procerebrum is modulatedby olfactory inputs to the tentacle (Gervais et al., 1996; Kimura etal., 1998b). To determine the primary mechanism for the frequencychanges of the LFP in the procerebrum, the tip, middle and basalregions of the digits of the superior and inferior tentacles wereelectrically stimulated and the LFP was recorded from theprocerebrum of Limax (Ito et al., 1999). Stimulation of the middleand basal regions of the digits of the inferior tentacle significantlydecreased the frequency of LFP whereas stimulation of the tipregion of the digits of the inferior tentacle and of all regions of thedigits of the superior tentacle increased the frequency of LFP.These findings suggest that the change in frequency of LFP in theprocerebrum depends on the excited region in the digits of thesuperior and inferior tentacles, providing the physiologicaldifferences in olfactory function between the superior and inferior

Fig.3. Inside structure of L. valentianus tentacles. D, digits; SE, sensoryepithelium; TG, tentacular ganglion.


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tentacles. These results may explain the previous behavioral dataconcerning the different roles of the superior and inferior tentacles(Suzuki, 1967; Cook, 1985; Chase, 1986; Friedrich and Teyke,1998; Kimura et al., 1998a). In particular, Kimura et al. showedthat the inferior tentacles play an important role in determining odorpreference, because the odor presentation to the inferior tentaclecan increase or decrease the frequency of LFP oscillations in theprocerebrum, depending on whether the odor is appetitive oraversive to the slugs (Kimura et al., 1998a). The mechanismunderlying this frequency change may be used by slugs that areconditioned to food odor.

To understand the neural circuits in the tentacles, LFP oscillationsin the digits and the tentacular ganglia were analyzed in Limax. Forexample, the changes in LFP oscillations were recorded from thetentacular nerves after odor stimulation to the sensory epithelium.Recordings from the inferior tentacular nerves (medial lip nerves)connected to the inferior tentacular ganglia and sensory epitheliashow spontaneous oscillatory activity, primarily at frequencies of0.6–6Hz (Ito et al., 2001). Similarly, ca. 1.5Hz oscillatory activitywas recorded from the neuropil region of the tentacular ganglion ofthe superior tentacle (Inokuma et al., 2002). Further, the applicationof various odors (garlic, carrot and rat chow) changed the oscillatoryproperties of tentacular ganglion recorded from the superiortentacular nerve in Limax (Ito et al., 2003a). For example, theappearance of high-amplitude spontaneous oscillations wasaccompanied by a decrease in odor-evoked spike activity in thesuperior tentacular nerve and the appearance of odor-evoked spikeactivity was accompanied by a decrease in the amplitude ofspontaneous oscillations, demonstrating a significant negativecorrelation between spontaneous oscillations and odor-evoked spikeactivity (Ito et al., 2003a). These results suggest that the intrinsicoscillatory activity contributes to olfactory processing in pulmonatesin a manner different from that reported in mammalian olfactorybulbs (Adrian, 1950).

To analyze the dynamics of odor-processing circuits in the digitsand tentacular ganglia, Ito et al. (Ito et al., 2004) studied the effectsof -aminobutyric acid (GABA), glutamate and acetylcholine –which are neurotransmitters present in the tentacular ganglion (Itoet al., 2003b) – on the circuit dynamics of the oscillatory network(s)in the superior and inferior tentacular ganglia. Application ofGABA to the cell masses decreased the tentacular nerve oscillationwhereas the application of glutamate and acetylcholine to the digitsincreased the oscillations recorded at the digits. These resultssuggest that there are two intrinsic oscillatory circuits that responddifferentially to neurotransmitters in the tentacular ganglion inLimax. However, no differences have been found in theresponsiveness to these neurotransmitters between the superior andinferior tentacles.

Recently, the metacerebrum of the cerebral ganglion has beenidentified as one of the central loci of tentacular inputs in anotherterrestrial slug, Incilaria (Makinae et al., 2008). The stimulation ofthe superior tentacular nerves activates the medial and lateral halvesof the medial neuropil region of the metacerebrum almost evenlywhereas the stimulation of the inferior tentacular nerves activatesthe lateral half of this region more strongly than the medial half.Makinae et al. also demonstrated that the activation of themetacerebrum precedes that of the procerebrum by ca. 50ms whena superior or an inferior tentacle is electrically stimulated,implicating the existence of at least two types of fibers withdifferent conduction velocities within both the tentacular andmedial lip nerves (Makinae et al., 2008). Together with the fact thatthe metacerebrum receives direct sensory inputs from the

mechanosensory neurons located in the sensory pad (Ierusalimskyand Balaban, 2010), the differential timing of the activation of themetacerebrum and the procerebrum may reflect the differentmodalities of the inputs, i.e. the mechanosensory and olfactoryinputs.

Which pair of tentacles is necessary for olfactory-aversionlearning?

We have gained an understanding of the physiological andmorphological differences between the superior and inferiortentacles. We thus turn our attention to the role of each pair oftentacles in olfactory-aversion learning. In previous studies, slugshave been easily conditioned using carrot juice and 1% quinidinesulfate solution as the CS and the US, respectively (Sahley et al.,1981; Nakaya et al., 2001; Matsuo et al., 2002). In theseconditioning experiments, the slugs crawl towards carrot juice putin their path. When the slugs touch the carrot juice, quinidinesulfate solution is applied to the mouth. The memory retention testis performed later with the carrot juice. As described below, weexamined the pole of each pair of tentacles by its amputation in theolfactory-aversion experiments.

A previous study showed that the amputation of inferior tentaclesafter conditioning degrades memory retrieval whereas theamputation of superior tentacles has no such effect, suggesting thatthe olfactory inputs from the inferior tentacles are important for theretrieval of aversive memory (Kimura et al., 1999). However,several aspects of that study needed to be evaluated again. We thusre-examined the olfactory ability of Limax following tentacleamputation (Yamagishi et al., 2008). The results showed thatmemory formation was not altered by the amputation of either ofthe pairs before or after olfactory-aversion learning; likewise, theodor sensibility of Limax was maintained. These data suggestedthat either pair of tentacles is sufficient for the acquisition andretrieval of aversive olfactory memory.

Although the data by Yamagishi et al. demonstrated that the twopairs of tentacles are functionally redundant with respect toolfactory-aversion learning (Yamagishi et al., 2008), do slugs havetwo pairs only in preparation for injury? Can each pair substitutefor the other’s function, whatever that may be? In Achatina, eachpair of tentacles serves a function in some tasks other thanolfactory-aversion learning: trail following is exclusivelydependent upon the inferior tentacles whereas orientation towardsdistant odor source depends on the superior tentacles (Chase andCroll, 1981). Further, in food-attraction learning in Helix, theacquisition of olfactory memory requires sensory inputs conveyedby the inferior tentacles whereas the recall of memory requiresintact superior tentacles, indicating the functional specialization ofeach pair of tentacles in appetitive learning (Friedrich and Teyke,1998).

Such inconsistencies with Yamagishi et al. (Yamagishi et al.,2008) might be explained by differences in the learning paradigmused in the studies, that is, aversive versus appetitive conditioning.Another explanation is that the manner of tentacle inactivation wasdifferent: Friedrich and Teyke acutely anesthetized either pair oftentacular epithelia with lidocaine (Friedrich and Teyke, 1998) andKimura et al. also ‘acutely’ amputated either pair of tentacles(Kimura et al., 1999) whereas Yamagishi et al. amputated eitherpair of tentacles 7days before the behavioral experiments(Yamagishi et al., 2008), allowing for functional compensation bythe remaining pair of tentacles, if any. Tentacle amputation alsoaffects the cautiousness of the slug. Slugs that have had theirsuperior tentacles amputated tend to be very cautious in




approaching any odorant sources (Kimura, 2000; Yamagishi et al.,2008). Such changes in behavior make it difficult to interpret thebehavioral data in learning experiments. However, the data ofYamagishi et al. at least demonstrated that slugs could manage toacquire and retrieve olfactory-aversion memory in the absence ofeither pair of tentacles (Yamagishi et al., 2008).

A pair of procerebraStructure and function of procerebra

The procerebrum is one of the loci that receive olfactory inputsfrom the tentacles. The procerebrum is a division of the cerebralganglion unique to terrestrial pulmonates and is specialized to theprocessing of olfactory information (Ratté and Chase, 1997; Rattéand Chase, 2000; Chase, 2000) (Fig.4). The number of neuronspresent in the combined left and right procerebra is estimated to beca. 105 in Limax, comprising approximately half of all the neuronsin the brain (Gelperin and Tank, 1990; Chase, 2000). The neuronsare small – 5 to 12m in diameter – and densely packed, with theirneuropils extending into the core region within the procerebrum.

The olfactory function of the procerebrum is indicated by thefact that it receives axons of the tentacular nerve and the medial lipnerve from the superior and inferior tentacular ganglions,respectively (Chase and Tolloczko, 1993; Kawahara et al., 1997;Ierusalimsky and Balaban, 2010). Procerebrum neurons alsoreceive direct inputs from olfactory sensory neurons, bypassing thetentacular ganglion (Chase and Tolloczko, 1993), indicating thatthe procerebrum serves not only as a secondary olfactory center butalso as a primary olfactory center. Recently, however, Ierusalimskyand Balaban reported that such a bypassing pathway is not observedin the olfactory ascending pathway in Helix and that few such directpathways are mechanosensory tracts (Ierusalimsky and Balaban,2010).

Injection of dyes into the cell bodies of procerebrum neuronsshowed that some cells possess long neurites that extend into the

contralateral side of the cerebral ganglion as well as into themesocerebrum in the ipsilateral cerebral ganglion (Ratté and Chase,1997; Ratté and Chase, 2000). Because these neurites have mostlyoutput synapses on their distal processes, whereas they have mostlyinput synapses near the cell body, the centrally projecting cellsprobably carry processed outputs from the procerebrum to motorcontrol neurons, including those responsible for food finding orlocomotion. In fact, some of the procerebrum neurons send outputsto the pedal ganglion as well as to the other regions of the cerebralganglion, and the membrane potential in these neuronssynchronizes with the LFP in the procerebrum (Chase andTolloczko, 1989; Ratte and Chase, 1997; Gelperin and Flores,1997; Shimozono et al., 2001).

The procerebrum consists of three layers: the cell mass, terminalmass and internal mass layers (Fig.5). The cell mass layer containsa large number of cell bodies of small neurons. The terminal masslayer and internal mass layer are neuropil layers. The procerebrumneurons receive inputs from the tentacular nerves at the terminalmass layer (Kawahara et al., 1997; Matsuo et al., 2010c). Theyalso receive serotonergic and Phe-Met-Arg-Phe-NH2ergic(FMRFamidergic) projections (Inoue et al., 2004; Kobayashi et al.,2010), as well as GABAergic projections (S.K., R.M. and E.I.,unpublished observation), in the terminal and/or internal masslayers in Limax, suggesting that the procerebrum is under complexneuromodulatory controls from the outside (Gelperin et al., 1993).

The procerebrum spontaneously produces a periodic slowoscillation of LFP (ca. 0.7Hz), even in the absence of olfactoryinputs (Gelperin and Tank, 1990). The LFP oscillation is producedby the concerted actions of two types of neurons that are thoughtto be the major constituents of the procerebrum. A small percentageof procerebrum neurons fire periodic bursts of action potentials(bursting neurons) (Kleinfeld et al., 1994; Watanabe et al., 1998).These bursting neurons are slightly larger than nonburstingneurons, which constitute the majority of neurons in theprocerebrum (Watanabe et al., 1998). The nonbursting neurons aresynaptically inhibited by the bursting neurons via glutamatergicinputs in Limax (Watanabe et al., 1999; Matsuo et al., 2009). Thepotential oscillations arise from the summed synaptic currents inthe nonbursting neurons triggered by inhibitory glutamatergicinputs from bursting neurons whereas the phase gradient along thelongitudinal length of the procerebrum is caused by differences inthe excitability of bursting neurons in the apical and basal regions(Watanabe et al., 2003).

Fig.4. Isolated central nervous system of L. valentianus without the buccalganglia showing the positions of the left procerebrum (PC), themetacerebrum and the mesocerebrum. A, anterior; L, left; P, posterior; R,right. Scale bar, 1.0mm.

Fig.5. Layer structure of the left procerebrum (PC) in L. valentianus(horizontal cross-section). CeG, cerebral ganglion; CM, cell mass layer; IM,internal mass layer; TM, terminal mass layer.


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The precise function of the procerebrum and the role of itsoscillations have been controversial. In some experiments,stimulation of a tentacle with odors produces changes inspontaneous oscillations, including amplitude and frequencyeffects (Gelperin and Tank, 1990; Gervais et al., 1996; Kimura etal., 1998b; Nikitin and Balaban, 2000; Cooke and Gelperin, 2001;Samarova and Balaban, 2007; Samarova and Balaban, 2009). Inaddition, odor stimulation causes a collapse of the phase gradientacross the procerebrum in the optical recordings; that is, theoscillations momentarily cease to propagate as a wave (Delaney etal., 1994; Gervais et al., 1996). Schütt et al. reported that severalodors each cause specific changes in the oscillations, with eachodor producing oscillations at a characteristic peak frequency(Schütt et al., 1999).

Despite all these reports of the effects of odor on spontaneousprocerebrum oscillations, suppression of the oscillations does notcompletely eliminate odor-evoked responses in efferent cerebralnerves (Teyke and Gelperin, 1999). This last result, with others, ledGelperin (Gelperin, 1999) to propose that the procerebrum isprimarily a learning machine, it is more involved in odordiscrimination than in odor recognition and the function of itsoscillations is to spatially segregate learned odor representations.Supporting this notion, Teyke and Gelperin (Teyke and Gelperin,1999) and Sakura et al. (Sakura et al., 2004) demonstrated thatsuppression of the procerebrum’s oscillatory activity by an inhibitorof nitric oxide synthase, which has been vigorously investigated inLimax (Gelperin et al., 1996; Gelperin et al., 2000; Fujie et al.,2002; Fujie et al., 2005; Matsuo et al., 2008; Matsuo and Ito, 2009),abolishes the ability to discriminate similar odors even while thedetection of odor itself remains intact in Limax. In many studies,the mere application of an odor did not cause a frequency change(Kimura et al., 1998a; Inoue et al., 2006; Samarova and Balaban,2009). It is only the odors that have some meaning to pulmonatesthat can elicit frequency changes in LFP oscillation; that is, onlyodors that the pulmonates approach or avoid can induce a frequencychange in the procerebrum. Samarova and Balaban recently showedthat the timing of the frequency change in the procerebrumcoincides with decision making (approach or escape) in freelymoving snails (Samarova and Balaban, 2009).

Memory locus of olfactory-aversion learningIn 2006, evidence suggested that the procerebrum is essential forolfactory-aversion learning in Limax (Kasai et al., 2006). In a seriesof olfactory-aversion learning experiments, the paired procerebrawere bilaterally lesioned 7days prior to conditioning. Most of thelesioned slugs did not avoid carrot juice (CS) in the memoryretention test performed 1day after the conditioning. That is, eitherthe acquisition or retrieval of memory was abolished by lesioningof the bilateral procerebra. The slugs whose procerebra werelesioned after conditioning also did not avoid carrot juice in thememory retention test, even if the procerebra were lesioned 7daysafter the conditioning. These results clearly demonstrate that theprocerebrum is a necessary component in the retention and/orretrieval of olfactory-aversion memory in Limax. However, Kasaiet al. also demonstrated that procerebrum lesion does not damageodor-sensing ability because procerebrum-lesioned slugs can avoidinnately aversive odorants such as garlic or onion and yet canapproach their everyday food (Kasai et al., 2006).

A subsequent investigation, using an experiment to exploit thespontaneous recoverability of the procerebrum from injury,revealed that the procerebrum is the storage site of olfactory-aversion memory and is not a mere conductor of neuronal

information about memory stored elsewhere (Matsuo et al., 2010a).Other experiments (Matsuo et al., 2010a) showed that the surgicallylesioned procerebrum spontaneously regenerates within a 1-monthrecovery period through neurogenesis from neuronal progenitorcells remaining within the procerebrum (Zakharov et al., 1998;Watanabe et al., 2008), and that the procerebrum substantiallyrecovers its volume. The regenerated procerebrum also restores theoscillatory activity of LFP. The slugs can achieve olfactory learningafter recovery but they cannot retrieve olfactory memory acquiredprior to surgery (Matsuo and Ito, 2008; Matsuo et al., 2010a). Theseresults suggest that surgical damage to the procerebrum irreversiblyextinguishes the memory stored in the procerebrum, and supportthe notion that the procerebrum is the storage site of olfactory-aversion memory (Gelperin, 1999).

However, to date it has been hypothesized that only the unilateralprocerebrum is used for olfactory-aversion learning. This isbecause the unilateral labeling of procerebrum neurons wasobserved after an injection of Lucifer Yellow into the body cavityfollowing conditioning in Limax (Kimura et al., 1998b; Ermentroutet al., 2001; Sekiguchi et al., 2010). Although the precise meaningof this band-shaped labeling is still elusive, Sekiguchi et al. recentlyproposed a model with two layered oscillators (neuropil and cellmass layers) to explain this phenomenon (Sekiguchi et al., 2010),and considered this band to be the most strongly depolarized regionduring conditioning by the synchronized activity in the two layers.However, their model did not refer to laterality in the labeling ofthe procerebrum.

Matsuo et al. recently presented data supporting the notion ofunilateral memory storage through unilateral procerebrum lesionexperiments (Matsuo et al., 2010b). The number of slugs with intactmemory performance was reduced by ~50% after the procerebrumwas surgically lesioned unilaterally before or after the conditioning.There were no differences in the memory performance of slugswhose right versus left procerebrum was lesioned. Those authors alsoshowed that there was no lateral memory transfer from oneprocerebrum to the other up to 7days after the conditioning. Theresults demonstrated that either the left or right procerebrum israndomly used for olfactory learning and memory storage, and thatthe side of use is determined at the level of the olfactory ascendingpathway to the procerebrum. This is explained by the fact that theunilaterally lesioned slugs can learn normally if the tentaclesipsilateral to the lesioned procerebrum are amputated at the sametime, indicating that there is some competitive mechanism betweenthe right and left olfactory inputs before they reach the procerebrum.

Unilateral tentacle amputation experiments (Matsuo et al.,2010b) suggested that the procerebrum ipsilateral to the remainingtentacle is always used for memory storage. These results alsosupport the view that the olfactory projection to the procerebrumfrom the tentacles is primarily ipsilateral (Chase and Tolloczko,1989; Matsuo et al., 2010c). In the presence of tentacles on onlyone side, the slugs inevitably use the procerebrum on the same sidefor memory acquisition and storage. This means that the slug brainsomehow monitors the state of olfactory inputs from both sides,and one side of the olfactory system can take over the mnemonicfunction in case the other side is functionally disabled.

It is uncertain how the brain decides which side of theprocerebrum to use for processing and storing memory in thepresence of bilateral tentacles. If this decision is made beforeolfactory inputs reach the procerebrum, the metacerebrum mightplay a role in this process because the timing of sensory inputsarrival to this region precedes that to the procerebrum (Makinae etal., 2008). Otherwise, lateral inhibition between the right and left




procerebra might result in the unilateral use of the procerebrum ina winner-take-all manner through an unidentified mechanism(Teyke et al., 2000).

ConclusionsMorphological and neuroethological analyses revealed that thesuperior and inferior tentacles differ from each other so that eachpair can function in optimized and specialized ways. In the case ofinjury, however, either pair can substitute for the function of theother in olfactory-aversion learning tasks. This might be enabledby the overall similarity in the structure and cellular componentsbetween the superior and inferior tentacles. Such flexibility is,however, restricted to the case when either tentacle is damaged,because selective damages to the unilateral procerebrum result inthe reduction of learning performance in that half withoutcompensation by the remaining procerebrum (Matsuo et al.,2010b). In this sense, only the tentacles have functional redundancyfor olfactory learning. This may be because the tentacles,appendages serving as multimodal sensory organs, are more proneto suffer from damage occurring in a pulmonate’s daily life incomparison to the procerebra, which are protected in the pulmonatebody. However, both the tentacle and the procerebrum have anexquisite ability to regenerate spontaneously after a recoveryperiod. We do not yet have a clear explanation why the pulmonatesuse only a unilateral procerebrum for mnemonic function everytime the slug learns. The doubling of memory capacity is onepossibility (Gelperin, 1999). Further, the existence of right and leftprocerebra may provide greater abilities to express tropotaxis byprocessing information conveyed from bilateral tentacles.

GlossaryCerebral commissures

Nerve bundles connecting the right and left cerebral hemiganglia ofpulmonates. Serotonergic, GABAergic, glutamatergic and dopaminergicnerves pass within these bundles (Matsuo et al., 2009; Makino and Yano,2010).

ChemotaxisAn animal’s directional movement strategy based on an external chemicalsubstance. An animal may move towards (positive chamotaxis) or awayfrom (negative chemotaxis) the chemical source.

Conditioned stimulusA chemical or physical stimulus that evokes a response acquired byPavlovian classical conditioning. Usually, a conditioned stimulus does nottrigger any response in an animal before learning. However, it evokes abehavioral response after the simultaneous (or temporally close)presentation of another stimulus (an unconditioned stimulus) that alwaysevokes that behavioral response.

KlinotaxisAn animal’s behavioral strategy during directional movement. Inklinotaxis, an animal determines its locomotive orientation by successivelysampling the external environmental cues, thereby monitoring its ownongoing movement. In contrast to tropotaxis, in which the animal monitorsspatial gradient of environmental cues, the animal monitors temporalchange in klinotaxis.

Local field potentialExtracellular electrical potential reflecting the membrane potential changesof the cells near the tip of a recording electrode. This potential change isproduced by the sum of the ionic currents passing through the membranesof the neighboring cells.

MetacerebrumA part of the cerebral ganglion of a terrestrial pulmonate. The metacerebrumis situated at the posterior part of the cerebral ganglion near thecerebropleural and cerebropedal connectives, and constitutes the major partof the cerebral ganglion. Motor neurons, sensory neurons and interneuronsare all contained in this structure (for details, see Chase, 2000).

PhototaxisAn animal’s directional movement strategy based on light. In positivephototaxis an animal moves towards a light source whereas in negativephototaxis an animal moves away from the light source.

ProcerebrumA specialized structure in the cerebral ganglion of a terrestrial pulmonateconsisting of ca. 105 small interneurons involved in olfactory informationprocessing. This structure is absent from aquatic pulmonates. Procerebralneurons receive direct and indirect olfactory inputs from tentacles andsend outputs to various brain structures including the pedal ganglia. Theprocerebrum is involved in higher olfactory functions such as odordiscrimination and olfactory learning.

TropotaxisAn animal’s behavioral strategy during directional movement. Intropotaxis, an animal determines its locomotive orientation by comparingthe sensory inputs received from bilateral sensory organs.

Unconditioned stimulusA chemical or physical stimulus that always evokes a behavioral responsein an animal. It has some significance for the animal’s survival in mostcases. Once an unconditioned stimulus is presented (in some casesrepeatedly) in combination with a neutral stimulus (a conditionedstimulus) that does not trigger any response, the animal learns that theconditioned stimulus is a predictive sign of the unconditioned stimulus,and the former evokes some response (a conditioned response) afterlearning.

AcknowledgementsThis work was supported by Grants-in-Aid for Scientific Research (KAKENHI)from the Japan Society for the Promotion of Science (JSPS) to R.M. (no.22570077) and E.I. (no. 21657022).

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