Associative Learning in Limax

Given the goal of understanding the cellular basis of associative learning (Kandel, 2001), comparative physiology seeks to identify a brain donor with highly developed associative learning and a nervous system well suited to biophysical analysis. Early work made clear that the central neurons and networks of gastropod mollusks were particularly favorable for the cellular analysis of synaptic plasticity and behavioral control. With this background we chose to explore the odor-learning ability of the terrestrial gastropod mollusk Limax maximus, which has large central neurons that are useful for studies of calcium transients during single-action potentials (Chang et al., 1974), neural control of heart rate (MacKay and Gelperin, 1972), cellular analysis of [³H]-2-deoxyglucose uptake (Sejnowski et al., 1980), neural control of feeding motor programs (Prior and Gelperin, 1977; Delaney and Gelperin, 1990c), and serotonergic modulation of feeding motor programs (Gelperin, 1981).

Learning cued by olfactory stimuli was chosen because olfactory systems in a wide variety of species can modify their input-output functions, linking odors to behaviors in ways that depend on previous olfactory experience. Synaptic plasticity and learning are essential components of olfactory information processing from mollusks to mammals (Hudson, 1999). Plasticity can be induced by passive exposure to odors or by arranging stimulus contingencies in which odors predict the presence or absence of other stimuli or rewards (Sahley, 1990; Eichenbaum, 1998; Slotnick et al., 2000). The ample evidence of plasticity in olfactory processing systems over wide phyletic boundaries suggested that Limax would be a good candidate for behavioral assessment of odor learning. I will emphasize here recent work in the Limax odor-learning system and related work on olfactory learning in closely related species—Helix, for example—which has appeared since my previous review (Gelperin, 1992).

Limax is an odor-dominated species dependent on olfaction for finding food, mates, and homesites (Gelperin, 1974). Associative learning about odor cues is rapid (Gelperin, 1975; Teyke, 1995) and shows several higher-order contingencies such as compound conditioning, second-order conditioning, and blocking (Sahley, 1990; Sekiguchi et al., 1991; Yamada et al., 1992; Sekiguchi et al., 1994; Suzuki et al., 1994; Sekiguchi et al., 1997). Appetitive conditioning can modify feeding behavior so that previously aversive odors become attractive (Sahley et al., 1992; Gelperin, 1999). The reliable and robust nature of odor conditioning in Limax, combined with the complex nature of the logic operations performed during odor conditioning (Gelperin et al., 1986) prompted an exploration of the central circuits for odor processing.

Central Olfactory Centers

Primary and second-order input from olfactory receptors projects to a distinctive integrative center, the procerebral (PC) lobe of the cerebral ganglion (Chase, 2000), where some 10⁵ interneurons process olfactory inputs (Ratté and Chase, 1997, 2000) and may store odor memories (Kimura et al., 1998a; Nakaya et al., 2001). Our initial search for the central site of odor learning therefore focused on the PC lobe, which has oscillatory dynamics of its local field potential (LFP) (Gelperin and Tank, 1990; Kawahara et al., 1997) and propagates activity waves along its apical-basal axis (Delaney et al., 1994).

Because odor-memory coding in the PC lobe depends on the dynamics of wave propagation, it is important to understand the mechanism of wave initiation and propagation. The PC lobe contains a small (1-2 percent) population of bursting inhibitory neurons (Watanabe, 1998) that seem to couple to each other by electrical and excitatory chemical synapses (Ermentrout et al., 2001). Two-photon laser-scanning microscopy has discerned two populations of inhibitory-bursting neurons, differing in the speed and direction of propagation of calcium-based action potentials in their neurites (Wang et al., 2001). The bursting inhibitory neurons produce chloride-mediated inhibitory synaptic potentials in the major population of nonbursting neurons (Watanabe et al., 1999). The population of bursting inhibitory neurons shows a gradient of excitability from the apex to the base of the PC, such that bursting occurs first in the most apical bursting neurons and then, because of excitatory coupling between bursting neurons, propagates along the apical-basal axis to the base of the PC lobe, where the activity wave ends. The gradient of excitability is shown by taking a series of transverse slices of the PC lobe along the apical-basal axis and measuring the frequency of spontaneous oscillations of the LFP in each slice. The most apical slice oscillates fastest, the most basal slice oscillates slowest, and the intermediate slices have intermediate oscillation frequencies, depending on their apical-basal position.

There is evidence of the apical-basal activity wave in two-site LFP measurements made simultaneously in apical and basal recording sites (Ermentrout et al., 1998; Gelperin et al., 2001) or in optical recordings where voltage-sensitive or calcium-sensitive dyes stain PC neurons. Sequential images of the PC lobe based on the calcium or voltage signals show the initiation of the activity wave at the apex and its propagation to the base (Kleinfeld et al., 1994; Toda et al., 2000; Nikitin and Balaban, 2001b). The LFP signal at a particular site along the apical-basal axis is produced by the simultaneous generation of a 5-7-mV inhibitory synaptic potential in a large number of nonbursting neurons by synaptic divergence from the local bursting neurons. The oscillatory nature of the LFP derives from the periodic nature of the activity wave in the bursting neurons propagating past a particular recording site along the apical-basal axis. Oscillatory dynamics is a universal feature of olfactory analyzers in mollusks, arthropods, and vertebrates (Tank et al., 1994; Gelperin, 1999; Laurent, 1999) and may contribute an essential temporal component to the odor code.

Odor-Memory Storage in the Procerebral Lobe

Learning-dependent labeling of a band of non-bursting PC neurons after one-trial odor conditioning provides some of the most direct evidence that odor memories are stored in the PC lobe (Kimura et al., 1998a; Gelperin, 1999). The learning-dependent labeling has been demonstrated as a consequence of both aversive and appetitive one-trial odor conditioning. The slug is given a single training trial with odor as a positive or negative conditioned stimulus, and then, twenty minutes after the conditioning trial, the animal receives an injection of with Lucifer yellow (LY) into the blood space. The hour-long interval that begins twenty minutes after the conditioning trial is when the short-term memory of odor conditioning is converted to a long-term form (Sekiguchi et al., 1991; Sekiguchi et al., 1994; Sekiguchi et al., 1997). The LY in the PC neuron somata after conditioning is contained in membrane-bound vesicles, as in the original reports of "activity-dependent" LY labeling in fly retina (Wilcox and Franceschini, 1984), perhaps because of an activity-dependent pinocytotic process. The causal coupling between electrical, synaptic, or biochemical events in the labeled neurons and pinocytotic uptake of LY is unknown.

The striking feature of the learning-dependent labeling in the Limax PC lobe is that various control procedures, such as odor presentation alone, which do not allow learning to occur, do not lead to LY labeling (Kimura et al., 1998a). If two odors are used as separate conditioned stimuli during training in the sequential aversive training trials, two LY-labeled bands appear in one PC lobe (Kimura et al., 1998a). The unilateral nature of the learning-dependent labeling was completely unexpected, but is also occurs in the replication and extension of the original work. (Gelperin, 1999) The existence of crossed inhibition between the right and left odor-processing circuits, demonstrated in an in vitro nose-brain preparation (Teyke et al., 2000), may explain why only one PC lobe is the dominant site of odor-memory storage.

The second major indication that the PC lobe is the likely site of odor-memory storage is that one-trial odor conditioning selectively activates a small set of genes in PC lobe neurons (Nakaya et al., 2001). Brain tissue from 200 Limax given one-trial odor conditioning was obtained, and differential mRNA expression was compared between this collection of learned brain and tissue from 200 brains of control animals given odor stimulation (CS) and an aversive unconditioned stimulus (US) with a CS-US delay too long to permit learning to occur (Gelperin, 1975). A gene coding for a twenty-three amino-acid peptide was identified, cloned, and sequenced and the deduced peptide was constructed for antibody production. The level of expression of the learning-activated gene was clearly enhanced selectively in neurons of the PC lobe in trained slugs relative to control slugs (Nakaya et al., 2001). The peptide is secreted to extracellular space and may play a role in stabilizing synapses, as suggested by recent work on cellular consequences of learning in Drosophila (Connolly and Tully, 1998).

Modulation of Procerebral Lobe Dynamics

The PC lobe has both intrinsic and extrinsic synaptic modulation using twenty-one known and putative neurotransmitters (Gelperin, 1999), notably nitric oxide (NO) (Gelperin, 1999), carbon monoxide (Gelperin et al., 2000), acetylcholine (Watanabe et al., 2001), dopamine (Gelperin et al., 1993; Rhines et al., 1993), serotonin (Yamane, 1989; Inoue, 2001), and glutamate (Watanabe et al., 1999), along with numerous small peptides such as FMRFamide and small cardioactive peptide B (Yamane and Gelperin, 1987; Cooke and Gelperin, 1988). It is therefore not surprising that recordings of PC lobe LFP in vivo using implanted fine-wire electrodes reveal a much richer range of activity than the isolated brain in vitro (Cooke and Gelperin, 2001). There are periods in vivo during which the 0.5-1.0 Hz oscillation of the PC LFP in vitro is recorded, but during other periods the diversity of LFP waveforms makes clear that the PC lobe has modes of activity in vivo not predicted from recordings in vitro.

The maintenance of LFP oscillations and therefore wave propagation is dependent on synthesis of NO in the Limax PC lobe. If NO synthesis is blocked in the terrestrial snail Helix by injecting a substituted arginine that blocks the activity of NO synthase, odor learning is blocked (Teyke, 1996). It is tempting to speculate that the dose of NO synthase blocker that blocks oscillatory dynamics in the PC lobe is also the dose that produces the odor-learning deficit. The Limax PC lobe stains intensely for nitric oxide synthase (Cooke et al., 1994; Gelperin et al., 2001). NO may set the level of bursting by the bursting neurons in the PC and hence determine the frequency of LFP oscillation and perhaps the rate of wave propagation (Inoue, 2001).

The use of olfactory nerve (ON) shock as a substitute for odor stimulation makes it is clear that odor inputs to the PC lobe have a phase-dependent effect on the bursting neurons (Inoue et al., 2000). Excitatory postsynaptic potentials (EPSPs) are recorded with short latency after ON shock in nonbursting neurons, while EPSPs with longer and variable latency are recorded in bursting neurons after ON shock. This procedure suggests a monosynaptic connection from fibers in the ON onto nonbursting PC neurons and an excitatory connection from nonbursting neurons onto bursting neurons. This direct excitation of non-bursting neurons by ON input is consistent with the known anatomy of the ON projection into the PC lobe as the ON fibers fill the neuropil of the PC lobe but do not project to the layer of neuronal somata (Gelperin and Flores, 1997; Kawahara et al., 1997). The neurites of the bursting PC neurons are confined to the layer of neuronal somata (Watanabe, 1998), so the bursting neurons cannot receive direct synaptic input from olfactory afferents. The demonstration that odor inputs can alter the frequency of the PC LFP (Gervais et al., 1996) must be due to odor inputs onto nonbursting neurons having indirect excitatory effects on bursting neurons.

Model of Odor Memory Formation

The current model of odor-memory band formation in the Limax PC lobe is summarized in Figure 1. The odor-memory bands have a bandlike shape because of the interaction of the wave front of the propagating-activity wave with the region of the PC most strongly driven by the odor used as the conditioned stimulus. The confluence of the activity wave-front and sensory drive from the odor produces a short-term memory of the odor. Behavioral evidence for the existence of a short-term odor memory comes from the finding that a brief odor stimulus can be given, and then, after tens of minutes, the delivery of unconditioned stimulus can still result in CS-US pairing (Gelperin, 1975). This long-delay or trace-conditioning aspect of odor conditioning is typical. The short-term odor memory is subsequently converted to a long-term odor memory because of the action of a modulatory transmitter liberated on the PC neurons storing the short-term memory as a consequence of application of the US.

We have elaborated the model to suggest that if two odors are learned, then the spacing of the two bands representing the odors will depend on the similarity of the two odors (see Figure 1). If the two odors are very similar (odor A and odor A'), then the odor-memory bands representing odor A and odor A' will be at the minimum interband spacing consistent with the ability to access the two memory bands individually and uniquely. If odor A is learned, if its odor memory band is formed, and then if wave propagation is blocked by blocking NO, then the brain will report that odor A' is the same as odor A (see Figure 1C). This result has been obtained with the isolated Limax nose-brain preparation (Teyke and Gelperin, 1999), whereas in honeybee blocking the oscillatory dynamics of the antennal lobe with picrotoxin produced the behavioral result that the bee could not discriminate between two similar odors but could discriminate between two very different odors (Stopfer et al., 1997). Blockade of NO synthase activity in honeybee antennal lobe impairs olfactory discrmination (Hosler et al., 2000).

PC Lobe Inputs and Outputs

Researchers have identified the nature of the behavioral modifications occurring during Helix food-odor conditioning and some of the motor pathways expessing the learned alteration in odor response (Peschel et al., 1996; Friedrich and Teyke, 1998). The same motor-output pathway that expresses odor learning in the Helix, the peritentacular nerves controlling the superior tentacle muscles, also expresses the modified motor output attributable to odor learning in the Limax (Teyke and Gelperin, 1999). Researchers have identified a neuron in the metacerebral lobe of the Limax PC lobe with a neurite in the PC lobe (Shimozone et al., 2001) that shows membrane potentials oscillations arising from input in the PC lobe. This neuron may convey PC-processed olfactory information to other chemointegrative sites in the cerebral ganglion, such as feeding command neurons.

The PC lobe receives inputs from other regions of the cerebral ganglion and from the tentacular ganglia adjacent to the olfactory receptor epithelia of the superior and inferior noses. The tentacular ganglia appear to have an oscillatory component to their LFP that is modulated by odor stimulation (Ito et al., 2001). The PC lobes contain neurites of neurons located in other parts of the cerebral ganglia (Ratté and Chase, 1997, 2000; Shimozone et al., 2001) and in the buccal (Gelperin and Flores, 1997) and pedal (Chase and Tolloczko, 1989) ganglia that provide the path-ways for integrating olfactory information with other inputs to make behavioral decisions. Activation of the PC-connected buccal neurons can reset the PC LFP oscillation, whereas the pedal cells are activated by PC stimulation but cannot reset the LFP oscillation by their activity. The PC LFP oscillation can also be modulated by electrical stimulation of the digits of the tentacular ganglia (Ito et al., 1999), a result that may be due to the FMRFamide contained in some of the primary sensory neurons projecting through the tentacular ganglia to the PC lobe (Suzuki et al., 1997).

Imaging Odor Memories

A number of imaging studies have attempted to clarify the nature of odor responses in the PC lobe (Kleinfeld et al., 1994; Inoue et al., 1998; Toda et al., 2000; Nikitin and Balaban, 2001a), particularly after odor conditioning (Kimura et al., 1998c; Nikitin and Balaban, 2000). Initial studies on naïve preparations showed that odor stimulation led to a collapse of the apical-basal phase gradient for a few cycles after odor stimulation (Kleinfeld et al., 1994). Imaging studies of PC lobe dynamics after one-trial odor conditioning have provided evidence for regional localization of odor excitation (Kimura et al., 1998c). Recordings of PC lobe LFP oscillations after odor training also indicate regional localization of baseline shifts in response to application of conditioned odors (Kimura et al., 1998b). It would be interesting to know if the region of localized excitation to a conditioned odor corresponds to the region of learning-dependent LY labeling in the same slug.

Summary

The olfactory-processing system of the Limax displays many of the design features of other species, including receptor turnover (Chase and Rieling, 1986), central neurogenesis (Zakharov et al., 1998), oscillatory dynamics of central-processing centers (Gelperin, 1999), rapid and long-lasting learning (Delaney and Gelperin, 1986), extensive local-feedback synapses (Zs.-Nagy and Sakharov, 1970; Ratté and Chase, 2000), glomerular processing of some inputs (Chase and Tolloczko, 1986), nitric-oxide-dependent synaptic plasticity (Teyke, 1996), memory-dependent alterations in early-processing stages (Nakaya et al., 2001), and several dozen neurotransmitters and neuromodulators involved in experience-dependent circuit reconfiguration (Gelperin, 1999). The comparative approach has produced evidence that molluscan model systems can yield principles of synaptic plasticity and associative learning (Kandel, 2001). Future work in the Limax will extend this foundation, for example, by clarifying how the biochemical substrates governing the transition from short-term memory to long-term memory (Burrell and Sahley, 2001; Sutton et al., 2001) are modified (Yin and Tully, 1996; Dubnau and Tully, 1998) to produce an odor-memory system wherein one-trial learning is the normal condition. Also, the learning-activated gene in the PC lobe has significant homology with gene sequences in zebrafish, mouse, and human, providing further support for the idea that molluscan model systems implement synaptic plasticity mechanisms of relevance to mammalian systems.

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