Gastropod Learning and Memory (Aplysia, Hermissenda, Lymnaea, and Others)
Summary and Keywords
Euopisthobranchia (Aplysia), Nudipleura (Tritonia, Hermissenda, Pleurobranchaea), and Panpulmonata (Lymnaea, Helix, Limax) gastropod mollusks exhibit a variety of reflex, rhythmic, and motivated behaviors that can be modified by elementary forms of learning and memory. The relative simplicity of their nervous systems and behavioral repertoires has allowed the uncovering of processes of neuronal and synaptic plasticity underlying non-associative learning, such as habituation, sensitization, and different forms of associative learning, such as classical and operant conditioning. Decades of work on these simpler and accessible animal systems have almost uniquely yielded an understanding into the mechanistic basis of learning and memory spanning behavior, neuronal circuitry, and molecules. Given the conservative nature of evolutionary processes, the mechanisms deciphered have also provided valuable insights into the neural basis of learning and memory in other metazoans, including higher vertebrates.
Learning is a process of acquiring new knowledge about the world and adapting one’s behaviors to the surrounding environment. In both vertebrates and gastropod mollusks, simple and complex behaviors can be modified by non-associative and associative learning procedures. Habituation and sensitization are two forms of non-associative learning by which animals learn from a single stimulus or independent stimuli (Thompson & Spencer, 1966). Habituation is a gradual decrement of a behavioral response to a repeated weak stimulus. This response can be restored to its initial strength and duration after application of a strong, often noxious stimulus, in a procedure of so-called dishabituation. Sensitization is an increase in a behavioral response induced by a single or repeated presentation of a noxious or appetitive stimulus. In addition to these relatively simple forms of learning, gastropod mollusks learn from an association of events. Such associative learning is based on two major and distinct paradigms: classical or Pavlovian conditioning and operant or instrumental conditioning. Pavlovian conditioning allows an animal to form a predictive relationship between two stimuli. Operant conditioning allows an animal to establish a predictive relationship between the expression of a specific behavior (so-called operant) and its positive or negative outcome. These two learning paradigms are well exemplified by Pavlov’s and Skinner’s experiments, respectively. In Pavlov’s experiments, conditioning in dogs was found to occur when a conditioned stimulus (CS), a bell tone, was temporally associated with an unconditioned stimulus (US), olfactory/visual stimuli from meat powder (Pavlov, 1927). After repetitive CS–US association, the CS alone, which initially does not trigger any salivary reflex, is eventually able to elicit this response (so-called conditioned response, CR) that is similar to the behavior evoked by the US alone. Skinner’s experiments illustrated that animals learn the rewarding or punishing consequences of their actions. In a box, a pigeon or rat eventually comes to press a lever (the operant) that leads to a reward delivery. While the animal learns this operant–reward association, the likelihood and temporal regularity of presses on the lever increase (Skinner, 1938).
Tracking the neuronal and synaptic basis of these elementary forms of learning and memory is extremely challenging in vertebrate nervous systems. However, since the late 1960s, remarkable behavioral and neurophysiological studies have been successfully conducted in gastropod mollusks and these simpler animals continue to provide fundamental knowledge of the neuronal substrates of learning and memory. Among Euopisthobranchia, Aplysia allows the comparative neuronal analysis of different forms of non-associative learning (habituation, dishabituation, and sensitization) and associative learning (classical and operant conditioning) in the same neuronal circuits for motor reflexes or motivated behaviors (Byrne & Hawkins, 2015; Hawkins & Byrne, 2015; Kandel, 2001). These studies have highlighted the molecular substrates of short-, intermediate-, and long-term memory induced by different learning paradigms. Among Nudipleura, Hermissenda has been successfully used to analyze visual integration and how classical conditioning modifies sensory and interneuronal pathways for phototaxic behavior. Pleurobranchaea and Tritonia have allowed analyzing plasticity of feeding or swimming motor pattern generators that is induced by non-associative or associative learning protocols. Among Panpulmonata, analysis of classical conditioning in Lymnaea illustrates that learning-induced plasticity can develop at multiple sites, including command-like neurons, in the circuit for feeding behavior. Lymnaea also serves to investigate operant conditioning-induced changes in the central pattern generator for aerial breathing. The roles of learning in olfactory processing and withdrawal behavior have been described in Limax and Helix. This article aims to provide an overview of these behavioral, neuronal, and molecular studies in Gastropods in order to highlight their essential contribution to understanding basic processes of learning and memory that subsequently have been found to be shared with more complex animals (Figure 1).
Habituation and Sensitization of Defensive Withdrawal Reflexes in Aplysia
Several forms of non-associative learning involving defensive withdrawal reflexes have been extensively studied using a reductionist approach in the marine sea slug Aplysia californica (Byrne & Hawkins, 2015). The wealth of scientific knowledge obtained from this work was recognized by the attribution of the Nobel Prize in Physiology or Medicine to Eric Kandel in 2000 (for review, see Kandel, 2001). In Aplysia, the external respiratory organ or gill is associated with an extensible tubular exhalant structure, called the siphon. A light touch to the siphon elicits a gill and siphon retraction into a cavity under the animal’s parapodia. This protective reflex is partly mediated by monosynaptic connections from sensory neurons that innervate the siphon, to the motor neurons responsible for the gill and siphon contraction. These siphon sensory neurons also activate polysynaptic pathways, including inhibitory, excitatory, and modulatory interneurons, which in turn regulate different aspects of the reflex (Frost & Kandel, 1995). Other reflexes, including the tail-withdraw reflex and the tail-elicited siphon-withdraw reflex, have also been examined. Studies of these pathways have provided essentially similar results about both their neuronal organization and the plasticity induced by learning and memory (Bristol, Sutton, & Carew, 2004; Byrne et al., 1991; Byrne & Kandel, 1996; Cleary, Byrne, & Frost, 1995).
A repeated harmless tactile stimulation applied on the siphon or the tail gradually decreases the magnitude and duration of the gill- and/or siphon-withdrawal reflex (Pinsker, Kupfermann, Castellucci, & Kandel,1970; Stopfer, Chen, Tai, Huang, & Carew,1996). This decrement, or habituation of the motor response, depends on features of the stimulus so that stimulus strength, interval of stimulus repetition within a series, or interval between series repetitions, determines the magnitude and persistency of this habituation. Moreover, consistent with dishabituation in vertebrates, habituation of the gill- and siphon-withdrawal reflex is restored by delivering a strong and novel stimulus to another body part of the animal (Pinsker et al., 1970).
At the circuit level, a habituation lasting < 30 minutes and implicating short-term memory formation is associated with a depression of the monosynaptic connections between the sensory and motor neurons, which are located in the abdominal ganglion. This short-term synaptic plasticity results from a gradual decrease of presynaptic neurotransmitter release (Castellucci & Kandel, 1974; Castellucci, Pinsker, Kupfermann, & Kandel, 1970). Long-term habituation, lasting > 24 hours and implicating long-term memory, is associated with a presynaptic depression of the sensory-motor connections, now correlated with a decrease in vesicle and active zone numbers (Bailey & Chen, 1983, 1988a; Carew, Pinsker, & Kandel, 1972). However, this depression also implicates postsynaptic changes in the motor neurons, which is associated with activation of glutamatergic receptors (AMPA and NMDA types), protein kinases, gene transcription, and protein synthesis. These molecular processes down-regulate expression of postsynaptic AMPA receptors that produce the excitatory postsynaptic potential in the motor neurons (Esdin, Pearce, & Glanzman, 2010; Ezzeddine & Glanzman, 2003).
Conversely, sensitization of the gill- and siphon-withdrawal reflex can be induced by a painful electric shock delivered to the animal’s tail (Carew, Castellucci, & Kandel,1971). This stimulus greatly enhances the capability of a weak siphon stimulation to elicit the withdrawal reflex. Whereas a single such noxious stimulus often produces short-term sensitization (STS), repeated and spaced shocks produce a long-term sensitization (LTS) of the reflex that lasts for days or weeks depending on the number of learning sessions and pattern of stimulations (Pinsker, Hening, Carew, & Kandel,1973).
The sensitizing tail shock activates a sensory-motor pathway that recruits several modulatory interneurons, such as the serotoninergic CB1 and the putative peptidergic/nitrinergic L29 interneurons, which modify the functional properties of the gill- and siphon-withdrawal reflex pathway (Antonov, Ha, Antonova, Moroz, & Hawkins, 2007; Hawkins & Schacher, 1989; Marinesco, Kolkman, & Carew, 2004). In behaving animals and in reduced neuronal preparations, sensitization training was found to increase the excitability of the sensory neurons and to strengthen the excitatory synapses between the sensory and motor neurons (Antonov, Kandel, & Hawkins, 2010). The synaptic facilitation is due to an increase in transmitter release from the sensory neurons (Castellucci & Kandel, 1976; Castellucci, Nairn, Greengard, Schwartz, & Kandel,1982; Kandel & Schwartz, 1982; Klein, Hochner, & Kandel, 1986), which is partly mediated by serotonin (5-HT) released during the tail shock (Brunelli, Castellucci, & Kandel,1976; Marinesco & Carew, 2002; Mercer, Emptage, & Carew,1991). This monoamine modulator binds to G protein-coupled receptors located on the membranes of the sensory and motor neurons and activates a type I adenylyl cyclase. This enzyme catalyzes the conversion of adenosine triphosphate (ATP) into cyclic adenosine-3-monophosphate (cAMP), which in turn activates a cAMP-dependent protein kinase (PKA) (Brunelli et al., 1976). Serotonin receptors also activate a phospholipase C and a calcium-dependent protein kinase (PKC) (Sugita, Baxter, & Byrne, 1994) that by phosphorylating various intracellular target molecules are able to modify the functional properties of the neurons and synapses (Byrne & Kandel, 1996; Castellucci et al., 1980).
Short-term facilitation of the sensory-motor connection is associated with a 5-HT-induced broadening of action potentials in the sensory neurons (Walters, Byrne, Carew, & Kandel, 1983), resulting from PKA/PKC-dependent phosphorylation of voltage-dependent potassium (K+) channels (Baxter & Byrne, 1990; Sugita et al., 1994). This spike broadening in the sensory neurons of the reflex pathway favors opening of voltage-dependent calcium (Ca2+) channels, increases transmembrane calcium influx, and consequently enhances Ca2+- dependent transmitter release (Hochner, Klein, Schacher, & Kandel, 1986). Closings/openings of several other K+ and Ca2+ channels by 5-HT and PKA-dependent phosphorylation also contribute to increasing membrane excitability and transmitter release from the presynaptic sensory neurons (Castellucci et al., 1980; Klein & Kandel, 1980; Siegelbaum, Camardo, & Kandel, 1982). In addition, a spike broadening-independent mechanism also contributes to short-term synaptic facilitation (Braha et al., 1990; Byrne & Kandel, 1996). In this process, a 5-HT and PKA-dependent phosphorylation of synapsin, a protein that binds synaptic vesicles to components of the cytoskeleton, promotes vesicular mobilization into readily releasable pools (Angers et al., 2002). All these data suggest that short-term sensitization and the related 5-HT-induced synaptic facilitation, or heterosynaptic facilitation, result from covalent changes in pre-existing proteins that favor transmitter release at presynaptic terminals.
LTS elicits changes at both pre- and postsynaptic sites and involves gene transcription and translation. It induces growth of new synaptic contacts and modifies some K+ currents, thereby increasing the intrinsic excitability in both the sensory and motor neurons (Bailey & Chen, 1988a, 1988b; Cleary, Lee, & Byrne, 1998; Scholz & Byrne, 1987). The cellular and molecular basis of LTS has been extensively studied in sensory and motor neurons dissociated from the nervous system and allowed to form monosynaptic connections in co-cultures. In analogues of LTS in cell culture, five spaced applications of 5-HT mimic the spaced electrical tail shocks. These 5-HT applications lead to a nuclear translocation of both PKA and mitogen-activated protein kinase (MAPK) in the sensory neurons (Martin et al., 1997). PKA promotes phosphorylation of cAMP-response element binding protein 1 (CREB1) and its subsequent binding to a regulatory region of the cAMP-response element (CRE) gene. The transcription factor CREB1 activates downstream molecular pathways responsible for promoting long-term membrane and synaptic plasticity (Bartsch, Casadio, Karl, Serodio, & Kandel, 1998; Dash, Hochner, & Kandel, 1990; Guan et al., 2002; Liu, Cleary, & Byrne, 2011). Conversely, MAPK inhibits binding of the memory repressor CREB2 to CRE. Thus, both molecular cascades converge synergistically to promote gene expression and long-term memory (Zhang et al., 2011). Long-term facilitation is associated with synthesis of ubiquitin hydrolase, an enzyme that favors PKA activity and the action of growth factors such as Neurotrophin and Tolloid/BMP-like protein (Chain, Schwartz, & Hegde, 1999; Kassabov et al., 2013; Zhang, Endo, Cleary, Eskin, & Byrne, 1997). New active synapses are stabilized by presynaptic local protein synthesis via the action of a self-perpetuating conformational change in prion-like protein cytoplasmic polyadenylation element-binding protein (CPEB) (Miniaci et al., 2008; Si, Choi, White-Grindley, Majumdar, & Kandel, 2010). Postsynaptic motor neuron changes are also implicated in long-term facilitation, such as a calpain-dependent cleavage of PKC to a persistently active kinase ApPKMIII, which is an isoform of PKMζ that is involved in memory consolidation in vertebrates (Bougie et al., 2009; Cai, Pearce, Chen, & Glanzman, 2011).
Epigenetic processes such as DNA methylation or histone acetylation are also necessary for the induction and maintenance of long-term memory and synaptic facilitation (Guan et al., 2002; Pearce, Cai, Roberts, & Glanzman, 2017). For example, evidence has shown that long-term memory, which is induced with five tail shocks, can be suppressed by a blockade of protein synthesis immediately after training. Despite this apparent loss of memory, long-term memory can be reinstated with an intermediate-term sensitization training consisting of three tail shocks only (Chen et al., 2014; Pearce et al., 2017). This truncated version of LTS training is not sufficient to induce long-term memory in previously untrained animals. Thus, despite the apparent memory loss, some molecular changes persist beyond the initial phase of protein synthesis. An intrahemocoelic injection of the DNA methyl transferase blocker RG-108, immediately before LTS training, blocks memory consolidation. Moreover, RG-108 injected after LTS training definitively erased long-term memory without any possible reinstatement with three tail shocks (Pearce et al., 2017). This finding therefore suggests the intriguing possibility that DNA methylation may persist after memory extinction and contribute to prime new experiences (Antzoulatos, Wainwright, Cleary, & Byrne, 2006).
In isolated ganglia, a prolonged application of 5-HT induces epigenetic silencing of CREB2. This process is mediated by a class of small non-coding RNAs (piwi interacting RNAs or piRNAs) which interact with the promoter region of the CREB2 gene (Rajasethupathy et al., 2012). In addition, 5-HT that induces memory down-regulates several small RNAs in clusters of sensory neurons. Among these RNAs, miR-124 and miR-22 negatively regulate expression of CREB1 and CPEB genes that promote long-term memory or synaptic facilitation (Fiumara et al., 2015; Rajasethupathy et al., 2009). In an experiment, total RNA from long-term sensitized animals was injected into naïve non-trained animals (Bédécarrats, Chen, Pearce, Cai, & Glanzman, 2018). Twenty-four hours later, these non-trained animals expressed a sensitized withdrawal reflex. Conversely, total RNA transfer from untrained to other untrained animals does not induce such a long-term memory. In cultured neurons from naïve animals, bath application of RNA from sensitized animals reproduces the increase in intrinsic excitability of sensory neurons as observed in LTS. Moreover, such “memory transfer” from trained donor to naïve animals is prevented by an intrahemocoelic injection of RG-108. Taken together, these results suggest that RNA can play a fundamental role in the induction of LTS via a mechanism requiring DNA methylation.
Intermediate-term memory lasting from 30 minutes to five hours can be expressed in addition to short- and long-term memory (Ghirardi, Montarolo, & Kandel, 1995; Sutton & Carew, 2000). Whereas short-term facilitation depends solely on presynaptic plasticity in sensory neurons, intermediate-term facilitation requires both pre- and postsynaptic mechanisms (Farah et al., 2017; Jin, Kandel, & Hawkins, 2011; Sossin, Sacktor, & Schwartz, 1994). Intermediate-term facilitation requires kinase activation and local protein synthesis as found in long-term facilitation. But unlike long-term facilitation, it does not require gene transcription. Intermediate-term facilitation is also associated with DNA and RNA methylation, which suggests common epigenetic processes in the intermediate- and long-term facilitation process (Yang et al., 2018).
Classical Conditioning of the Gill- and Siphon-Withdrawal Reflex in Aplysia
In addition to non-associative learning, Aplysia’s gill- and siphon-withdrawal reflex can be modified by classical conditioning, a simple form of associative learning (Carew, Hawkins, & Kandel, 1983; Carew, Walters, & Kandel, 1981; Hawkins & Byrne, 2015). In this paradigm, the CS is a light touch of the siphon that elicits a weak withdrawal response. The US is a noxious electrical tail shock identical to the stimulus used in the sensitization paradigm. In a classical conditioning protocol, the CS and US are paired so that the CS starts shortly before and persists during the US. After 15 such CS–US pairings, the CS alone is able to elicit a CR that resembles the motor response evoked by the US alone (i.e., a strong and long-lasting retraction of the gill and siphon). This paired procedure produces long-term memory as indicated by the persistence of the CR for several days after training. Control procedures in which the same number of random or unpaired presentations of the CS and US, or the presentation of CS or US alone, do not produce such an increase in the reflex strength. Moreover, Aplysia can learn from a differential conditioning procedure in which two CS are applied to different sites on the animal, the siphon and the mantle shelf. One of these stimuli (CS+) is paired with US, whereas the other (CS-) is explicitly unpaired with the US. After learning, only the CS+ elicits the CR. Because the CS+ and CS- sensory pathways converge onto the same motor neuron pool, this stimulus specificity that is revealed by differential conditioning suggests that learning is partly encoded in the sensory neurons belonging to the CS+ pathway.
The cellular and synaptic basis of classical conditioning have been investigated in reduced preparations that converse the abdominal, pleuropedal, and cerebral ganglia with their connective and peripheral nerves left attached to the tail and siphon (Antonov, Antonova, Kandel, & Hawkins, 2001, 2003). Electrophysiological studies in these preparations demonstrated that classical conditioning increases the magnitude of excitatory postsynaptic potentials (EPSP) that are elicited by the CS alone in identified LFS motor neurons. Part of this response results from a pairing-specific increase in magnitude of the monosynaptic EPSP evoked by action potentials in the LE sensory neurons. In addition, classical conditioning increases intrinsic excitability of the sensory, inter-, and motor neurons in the CS pathway (Antonov et al., 2003; Yang et al., 2015). This membrane and synaptic plasticity is partly induced by 5-HT, a modulator also involved in sensitization, and nitric oxide (NO) (Antonov et al., 2003, 2007). In classical conditioning, however, 5-HT modulation requires a concomitant activation of the CS pathway to induce the pairing-specific neuronal plasticity. Thus, an experimental depolarization of LE sensory neurons to mimic the effect of CS, when associated with the US, can reproduce the pairing-specific increase of the sensory-motor synaptic strength and sensory neuron excitability (Antonov et al., 2003; Buonomano & Byrne, 1990; Hawkins, Abrams, Carew, & Kandel, 1983; Walters & Byrne, 1983). Moreover, in reduced preparations, an association of the sensory neurons’ electrical activity with bath application of 5-HT or NO reproduces aspects of the neuronal plasticity that is induced by classical conditioning (Ocorr, Walters, & Byrne, 1985; Antonov et al., 2007). Thus, learning partly results from an activity-dependent and pairing-specific amplification of the neuronal plasticity induced by the sensitization protocol. In this activity-dependent modulation, adenylyl cyclase acts as a molecular coincidence detector that is essential in associative learning. This enzyme is activated by both the US-related 5-HT release and the CS or resulting Ca2+ influx through voltage-dependent Ca2+ channels that open in the presynaptic terminals. Consequently, the presynaptic production of cAMP is considerably amplified, leading to pairing-specific neuronal plasticity (Abrams, 1985; Abrams, Karl, & Kandel, 1991; Antonov et al., 2007; Eliot, Hawkins, Kandel, & Schacher, 1994).
In addition to this activity-dependent modulation, evidence indicates that classical conditioning-related plasticity at the sensory-motor synapse involves a Hebbian mechanism. According to Donald Hebb’s postulate, neuronal plasticity can emerge from coincident activity in pre- and postsynaptic neurons (Hebb, 1949). Such Hebbian plasticity was initially identified in co-cultures of sensory and motor neurons, and later found to be involved in classical conditioning (Antonov et al., 2003; Lin & Glanzman, 1994a, 1994b; Murphy & Glanzman, 1997). In this mechanism, both a CS-induced presynaptic glutamate release from the sensory neurons and a concomitant US-elicited postsynaptic depolarization are necessary to activate NMDA-type glutamate receptors in the motor neurons. Postsynaptic Ca2+ influx through these receptors activates kinases and induces long-lasting changes in AMPA-type glutamate receptors. These changes produce a long-term potentiation (LTP) of the sensory-motor connections, similar to hippocampal LTP in mammals. Thus, NMDA receptors are a second molecular detector of the CS–US association, which may operate in tandem with activity-dependent modulation. LTP-related postsynaptic changes are likely to activate a retrograde signal that further amplifies cAMP-dependent presynaptic cascades responsible for long-term facilitation (Bao, Kandel, & Hawkins, 1998; for discussion, see Lechner & Byrne, 1998).
Classical Conditioning of Feeding Behavior in Aplysia
In addition to sensory-motor reflexes, motivated behaviors, such as feeding behavior, can be modified by classical conditioning paradigms in Aplysia (Colwill, Goodrum, & Martin, 1997; Lechner, Baxter, & Byrne, 2000a, 2000b). Specifically, learning-induced changes in movement cycles of the tongue-like radula have been the most thoroughly studied because they are easily quantifiable and the neuronal networks that generate these movements have been identified in terms of constituent neurons and their synaptic connections (Cropper et al., 2004; Kupfermann, 1974; Nargeot & Simmers, 2012). The movement cycles are composed of a protraction phase immediately followed by a retraction phase. In addition, the two halves of the radula can close to grasp food. Depending on the phase relationship of closure during the protraction–retraction cycle, several radula movement patterns can be distinguished. In an ingestion cycle, closure occurs mostly during retraction, thereby drawing food into the buccal cavity. In an egestion behavior, closure occurs during the protraction phase, thereby expelling non-edible particles from the buccal cavity. These behaviors can be produced spontaneously or in response to sensory stimuli.
In a classical conditioning procedure, a tactile stimulus applied to the lips was used as a CS, while food was used as a US (Lechner et al., 2000a). The CS alone produces no or few ingestion behavioral cycles. However, after repeated CS–US pairing, the number of ingestion cycles elicited in response to the CS alone is considerably increased. This training paradigm induces long-term memory with a CR retention lasting more than 24 hours. In contrast, control animals do not form memory in response to unpaired presentations of the CS and US or to the US alone. The US is produced by the presence of food in the buccal cavity and the sensory signals are conveyed by the esophageal nerves that contain dopaminergic fibers.
Neuronal correlates of classical conditioning were investigated after training in isolated preparations of the cerebral and buccal ganglia (Lechner et al., 2000b). In such preparations from trained and control animals, the CR was elicited with brief electrical stimulations of a lip nerve. This stimulation triggers more ingestion motor patterns in preparations from trained than from control animals. This pairing-specific plasticity is specifically associated with an increase in the CS-evoked synaptic drive to the neurons B31/B32 in the network generating radula motor patterns.
An in vitro analogue of classical conditioning was also developed in which an electrical stimulation of the lip nerve was used as a CS and an electrical stimulation of the esophageal dopaminergic input nerve was used as a US (Kabotyanski, Baxter, & Byrne, 1998; Mozzachiodi, Lechner, Baxter, & Byrne, 2003; Reyes, Mozzachiodi, Baxter, & Byrne, 2005). This analogue experimental condition reproduces the pairing-specific increase in the number of CS-elicited ingestion motor patterns associated with an increase in the strength of the synaptic drive to B31/32 neurons. A pairing-induced increase in synaptic drive from the lip nerve to a cerebral-buccal interneuron (CBI-2), which in turn excites B31/32 neurons, was also reported. Finally, a commensurate decrease in membrane excitability and an increase in the strength of presynaptic inputs was found in B51, a neuron of the radula pattern generating network that is critically involved in the expression of the ingestion pattern (Lorenzetti, Mozzachiodi, Baxter, & Byrne, 2006). Overall, this neuronal plasticity, at least partly induced by dopamine, favors the transmission of neuronal activity in a sensory-motor pathway, thereby allowing the CS to trigger the CR.
Operant Conditioning of Feeding Behavior in Aplysia
Operant conditioning has been used to modify several behaviors including head weaving, gill- withdrawal, and feeding in Aplysia californica and/or Aplysia fasciata (Brembs et al., 2002; Cook & Carew, 1986, 1989a, 1989b; Hawkins, Clark, & Kandel, 2006; Lyons, Rawashdeh, Katzoff, Susswein, & Eskin, 2005; Nargeot, Petrissans, & Simmers, 2007; Schwarz, Markovich, & Susswein, 1988; Susswein & Schwarz, 1983; Susswein, Schwarz, & Feldman, 1986). Among these studies, analysis of behavioral changes in feeding behavior yielded fundamental insights into the neuronal basis of appetitive and aversive forms of this learning paradigm. Moreover, descriptions of the underlying neuronal circuits, which are also modified by classical conditioning (see section “Classical Conditioning of Feeding Behavior in Aplysia”), has provided the basis for comparative studies of how different forms of associative learning modify the same neuronal substrates for motivated behaviors.
In a form of aversive operant conditioning, animals learn that food is inedible (Lyons et al., 2005; Susswein et al., 1986). During the consummatory phase of feeding behavior, Aplysia grasps food with its radula and generates a swallowing act that leads to food entry into the buccal cavity. This operant can be long-lastingly modified by wrapping food in an inedible rough net. Then, stimuli arising from failed attempts to ingest food act as a punishment for expression of the ingestion movement cycles. Consequently, as conditioning progresses, the number of this operant decreases. Memory of this conditioning can be expressed from 15 minutes up to 24 hours depending on the training procedure. Long-term memory requires activation of protein kinases A and C, activates polyADP-ribosylation of nuclear proteins, and is likely to increase the expression of several genes and protein synthesis (Cohen-Armon et al., 2004). One locus responsible for these changes is in the buccal ganglia, which contain the radula motor pattern generating network. Moreover, punishment in this aversive conditioning is associated with a release of NO and histamine in the cerebral ganglia, which is connected to the buccal ganglia via the cerebro-buccal connectives. However, these transmitters may not act as simple punishers themselves, but rather they are likely to recruit an as yet unknown punisher to be effective (Katzoff, Ben-Gedalya, & Susswein, 2002; Katzoff, Miller, & Susswein, 2009;Katzoff et al., 2006). Interestingly, learning and memory are also modulated by different factors such as the presence of other species members, or the circadian clock and sleep cycles (Lyons et al., 2005; Schwarz & Susswein, 1992). Thus, greater memory formation is expressed in the diurnal Aplysia californica when training occurs in the daytime rather than during the night. In contrast, noctural Aplysia fasciata expresses better memory when trained during the night than during the day. Moreover, as in vertebrates, sleep-like rest in Aplysia can contribute to memory consolidation after operant conditioning (Vorster & Born, 2017)
Appetitive forms of operant conditioning of feeding behavior have also been investigated (Brembs et al., 2002; Nargeot, Petrissans, & Simmers, 2007; Susswein et al., 1986). In hungry Aplysia, the ingestive movement cycles of the radula can be expressed spontaneously or in the presence of a constant arousing food stimulus. A striking feature of these movement cycles is the variability of their expression, which is partly manifested by irregular time intervals between their occurrences. In an operant conditioning procedure, each emission of a radula movement cycle was associated with a reward composed of an ingestible piece of food, or food extract, which is experimentally introduced into the buccal cavity. Consequently this operant–reward association strongly increases the likelihood and regularity of emissions of the ingestion movement cycles. This more frequent and regular, compulsive-like, behavior is memorized for several hours, and is not expressed when the reward is not delivered in time with the spontaneous emission of the operant or when the reward is replaced by non-palatable elements (Nargeot et al., 2007). Thus, these behavioral changes are strictly dependent both on the association that characterizes operant conditioning and on the appetitive value of the reward.
Food reward delivery triggers bursts of action potentials in the dopaminergic esophageal nerves. On this basis, a learning paradigm analogous to brain self-stimulation that underlies operant learning in vertebrates has been developed in Aplysia. In this protocol, an electrical stimulation of the dopaminergic esophageal nerve was used as a reward equivalent that was delivered upon the expression of each ingestion movement cycle (Brembs et al., 2002). This associative paradigm strongly increases emissions of the operant as found after operant conditioning. Furthermore, the behavioral adaptation is not induced with a “yoked”-control paradigm in which the nerve stimulations are delivered in one animal depending on the operant expressed in another animal. This led to the conclusion that operant conditioning critically depends on an association of activity from a dopaminergic reinforcement pathway, with the ongoing activity of a central neuronal network responsible for generating a behavior.
Neuronal correlates of operant conditioning have been analyzed in isolated buccal ganglia from trained and control animals. These ganglia contain the central pattern generator (CPG) that expresses the radula motor patterns. This CPG network organizes two major decision-making processes that contribute to the variability in emission of the radula motor patterns (Nargeot & Simmers, 2012). One of these processes determines when to instigate a radula motor pattern, whereas the second one selects which motor pattern has to be expressed. Specifically, the first decision-making process concerns coordinated bursting activities in a small subset of interneurons (B30, B65) that are electrically coupled with an essential pacemaker neuron (B63) of the CPG network. In preparations from control naïve animals, these neurons generate successive bursts of action potentials and associated radula motor patterns at a low frequency and with irregular time intervals, similar to the expression of actual radula movement cycles in the intact animal. The second process is partly determined by the recruitment of B51 neuron, whose activity designates the pool of motor neurons appropriate for expression of the ingestion pattern (Nargeot, Baxter, & Byrne, 1999a, 1999b). During motor pattern genesis in preparations from control naïve animals, this B51 generates either a plateau with action potentials per cycle or only a subthreshold membrane depolarization. These two functional states of B51 lead to the expression of either ingestion or egestion patterns, respectively. Appetitive operant learning considerably increases the excitability and strength of electrical synapses in the B63, B30, and B65 neurons (Nargeot, Le Bon-Jego, & Simmers, 2009), which in turn contribute to difference aspects of the motor changes induced by learning. The increase in neuronal excitability underlies the learning-induced increase in frequency of motor pattern genesis, whereas strengthening the electrical synapses within this subset of pacemaker neurons is critical for inducing regular pacemaker activity that leads to stereotyped rhythmic motor pattern production (Sieling, Bédécarrats, Simmers, Prinz, & Nargeot, 2014). Operant learning also strongly increases the excitability of the B51 neuron, thereby increasing its functional recruitment within the CPG network and the likelihood of ingestion rather than egestion pattern occurrences (Brembs et al., 2002; Mozzachiodi, Lorenzetti, Baxter, & Byrne, 2008; Nargeot et al., 1999b). Together, this membrane and synaptic plasticity involved in the two essential decision-making processes transforms a variable, infrequent spontaneous behavior into a frequent and regularized rhythmic routine.
The operant conditioning-derived plasticity is partly mediated by monosynaptic connections from the dopaminergic esophageal fibers to the decision-making neurons. In in vitro neuronal preparations, induction of the neuronal plasticity is blocked by dopamine receptor antagonists (Bédécarrats, Cornet, Simmers, & Nargeot, 2013; Nargeot et al., 1999c), indicating that dopamine in Aplysia contributes to rewarding process as described in vertebrates. A single cell analogue of the operant learning was also developed, in which a puff of dopamine was used as reinforcement and an experimentally triggered plateau potential in a cultured B51 neuron was used as an analog of the operant (Brembs et al., 2002). This paradigm, which reproduces the operant–reward association in B51, increases B51 excitability and the likelihood of plateau potential generation as seen after behavioral training in vivo. This suggests therefore that the reward–operant association characteristic of operant conditioning can be encoded at the single cell level. An analysis of the association detection at this cellular level was also performed (Lorenzetti, Baxter, & Byrne, 2008). The findings indicated, firstly, that dopamine contributes to the association by activating a cAMP-PKA dependent signaling cascade via D1 receptors, while secondly, plateau potential generation in B51 induces a calcium influx that activates PKC. Detection of the association that results from the synergistic actions of these two molecular pathways was localized at the adenylyl cyclase level. This dual activation of this enzyme considerably increases cAMP production to a level necessary to modulate the neuronal properties of the decision-making neuron.
It is interesting to note that neuron B51 is a locus of plasticity not only for operant conditioning but also for classical conditioning. On the basis of this observation, a comparative study of the plasticity induced by appetitive forms of these two essential associative learning procedures has been performed (Baxter & Byrne, 2006; Brembs et al., 2004). This comparison showed that the same monoamine (i.e., dopamine) contributes to the reinforcement and induction of the underlying plasticity through an activity-dependent modulation process in the two forms of learning, both of which modify the same target decision neuron (i.e, B51). However, whereas operant conditioning increases B51’s intrinsic excitability, classical conditioning decreases it. Besides, classical conditioning increases the strength of the sensory-elicited synaptic drive to the feeding CPG, a plasticity that so far has not been reported for operant conditioning. Accordingly, whereas operant learning seems to favor autonomous processes of decision-making in the CPG, classical learning appears rather to weaken these processes and favor a synapse-driven conveyance of electrical activity from sensory inputs to motor output.
Non-Associative Learning of Escape Swim in Tritonia
Tritonia diomedea provides an interesting model for investigating how short-term non-associative memory is encoded in a large network. Tritonia’s escape swim consists of repeated body movement cycles elicited in response to a noxious stimulus. These cycles consist of a series of alternating ventral/dorsal body flexions, which are generated by a neuronal circuit that has been partly identified (Frost & Katz, 1996; Getting, 1989). When repeatedly elicited, this behavior expresses a sensitization of the latency to the post-stimulus onset of swimming (Brown, Frost, & Getting, 1996; Frost, Brandon, & Mongeluzi, 1998). The sensitization also lowers escape swim threshold. A neuronal analogue of this sensitization was developed in an in vitro brain preparation that contains the swim network and in which electrical stimulation of the peripheral pedal nerve 3 is used as a sensitizing stimulus (Brown, 1997; Hill, Vasireddi, Wang, Bruno, & Frost, 2015). Experiments using a voltage-sensitive dye to image the activity of several dozen neurons in the pedal ganglia showed that sensitization involves a rapid functional expansion of the escape swim network (Hill et al., 2015). In this process, the number of reliably bursting neurons participating in the swim motor pattern gradually increases with repeated nerve stimulation in correlation with the decrease in the response onset latency. These newly recruited neurons belong to a pool of non-bursting or variably bursting neurons that do not initially participate, or participate but not in all cycles, in the motor swim pattern. This sensitization-induced neuronal recruitment is associated with an intrinsic serotoninergic modulation (Hill et al., 2015; Katz, Getting, & Frost, 1994). An experimentally evoked tonic firing in the serotoninergic DSI neurons in the swim CPG mimics aspects of sensitization. Moreover, bath application of serotonin reduces the response onset latency and induces the network expansion. DSI neurons and serotonin produce and maintain sensitization-related memory, possibly through changes in neuronal excitability and synaptic strength (Katz et al., 1994; Katz & Frost, 1997).
Classical Conditioning of Phototaxic Locomotion in Hermissenda
The sea slug Hermissenda crassicornis was one of the first animal models used to investigate the neuronal basis of aversive classical conditioning (Crow & Alkon, 1978). Light elicits a phototaxic behavior that consists of locomotor movements toward light. In contrast, turbulences caused by waves force the animal to cling to the substrate. This adaptive behavior is immediately relevant for the animal’s survival in its natural benthic environment. In a classical conditioning paradigm, a flash of light (CS) is paired with turbulences induced by the orbital shaking (US) of the animal’s tank. After learning, light alone (CS) induces a foot shortening and inhibits the phototaxic behavior. Memory for this associative learning lasts from several minutes to weeks depending on the number of CS–US pairings.
The neuronal plasticity associated with this learning and memory has been investigated in isolated nervous preparations. Two types of photoreceptors (2 type A and 3 type B) in the animal’s eyes respond to the CS, with the type B inhibiting the type A receptors. In addition, both receptor types project their axons to the cerebropleural ganglia via direct monosynaptic and polysynaptic excitatory and inhibitory connections with several groups of interneurons. Electrical activity in these postsynaptic interneurons can trigger neuronal responses in the pedal motor neurons that contribute to locomotion. The US is detected by statocysts, a bilateral pair of gravity detecting organs. Statocyst hair cells play a role similar to the vestibular hair cells in vertebrates. These specialized sensory cells make polysynaptic axonal projections onto pedal motor neurons responsible for foot shortening (Crow, Jin, & Tian, 2013; Crow & Tian, 2004). The photoreceptors are a first and key site of convergence for the CS and US (Crow & Alkon, 1980). Specifically, the US pathway is composed of monosynaptic GABAergic synapses from the hair cells with the photoreceptors and of polysynaptic connections releasing 5-HT with the type B photoreceptors. Through these pathways, a depolarization of the photoreceptors (CS) paired with statocyst activation (US) causes a long-lasting increase in type B photoreceptor intrinsic excitability and a decrease in type A photoreceptor excitability (Alkon, Lederhendler, & Shoukimas, 1982; Crow & Alkon, 1978; Farley & Alkon, 1982). Learning also strengthens inhibitory synapses made by type B receptors with type A receptors (Frysztak & Crow, 1993, 1994). A second site of convergence in the CS and US pathways is composed of subsets of interneurons that receive excitatory or inhibitory synaptic inputs from the type B photoreceptors and statocyst hair cells. Classical conditioning induces a combination of changes in synaptic strength and intrinsic excitability in these interneurons (Crow & Tian, 2002, 2003). Accordingly, expression of the conditioned response not only results from learning-induced changes in visual sensory processing, but also from the interneuronal computations that determine the flow of information from sensory to motor neurons.
It is remarkable that the learning-induced plasticity of intrinsic excitability in the sensory cells involves an activity-dependent modulation similar to that described for associative learning in Aplysia. In Hermissenda, however, this process involves a persistent activation of PKC that is induced by a dual process: (a) a rise in intracellular calcium resulting from photoreceptor activation, associated with (b) an activation of phospholipase A2 and the production of arachidonic acid, which are induced by the release of GABA from the hair cells and its binding to GABAB receptors on the photoreceptor membrane. The ensuing PKC activation modifies the permeability of ion channels that in turn determine photoreceptor excitability (Farley & Auerbach, 1986; Farley & Schuman, 1991; Matzel, Lederhendler, & Alkon, 1990). Serotonin-induced activation of the MAPK signaling pathway in the photoreceptors also contributes to Pavlovian conditioning (Crow, Xue-Bian, Siddiqi,Kang, & Neary, 1998).
Aversive Learning of Feeding Behavior in Pleurobranchaea
The carnivorous Pleurobranchaea californica can be trained with classical conditioning to avoid a food stimulus (CS) that is paired with an electrical shock (US) (Mpitsos & Collins, 1975). The CS pathway includes command-like interneurons in cerebropleural ganglia that are essential for initiating feeding responses. These interneurons project to a half-center CPG located in the buccal ganglia that generates alternating cycles of radula protraction and retraction (Jing & Gillette, 2000; London & Gillette, 1986). Studies in semi-intact preparations indicated that the CS–US pairing increased a polysynaptic inhibition of the command neurons, thereby blocking expression of the radula protraction phase. This inhibition results from an increase in the intrinsic excitability of identified cerebropleural I2 inhibitory interneurons.
Classical Conditioning of Feeding Behavior in Lymnaea
Feeding behavior in the freshwater snail Lymnaea stagnalis can be modified by both appetitive and aversive classical conditioning procedures (Benjamin, Staras, & Kemenes, 2000). Importantly, the neuronal circuit generating feeding responses is well known, thereby allowing a detailed cellular analysis of classical conditioning at different levels in the network.
In response to a food stimulus, Lymnaea generates cyclic rasping movements of the radula that are composed of protraction, retraction or rasp, and swallow phases. The functional connectivity diagram of the feeding CPG generating these cycles has been established (Elliott & Benjamin, 1985). Each phase is produced by a specific class of interneurons in the buccal ganglia: N1 for protraction, N2 for retraction, and N3 for swallowing, that in turn activate dedicated pools of buccal motor neurons. Lip sensory neurons make synaptic connections with several higher-order command-like neurons in the cerebral ganglia. These high-order neurons consist of modulatory cerebro-buccal interneurons (CBI) with axonal projections to the buccal CPG. They include the serotoninergic cerebral giant cells (CGC) and the cerebral ventral interneurons 1 (CV1).
Appetitive classical conditioning procedures consist of pairing a CS, either a neutral amyl acetate application or a tactile touch onto the lips, with a US consisting of a sucrose application to the lips (Audesirk, Alexander, Audesirk, & Moyer, 1982; Staras, Kemenes, & Benjamin, 1999). After several CS–US pairings, trained animals express enhanced feeding responses to the CS alone, compared with animals that receive random or unpaired stimuli. Importantly, the strength and duration of appetitive learning depends on variables such as the age and motivational state of animals. For example, in young and food-deprived animals, a single CS–US association is sufficient to induce long-term memory lasting up to two weeks (Alexander, Audesirk, & Audesirk, 1984).
The cellular and molecular mechanisms for memory formation and consolidation have been investigated in semi-intact preparations and in isolated nervous preparations consisting of both the cerebral and buccal ganglia. The modulatory CBIs are loci for memory storage. Specifically, CGC and CV1 neurons were found to be major sites of non-synaptic plasticity involved in the classical conditioning. Learning induces a persistent depolarization of resting membrane potential in these neurons, which makes them more likely to fire action potentials in response to the CS (Jones, Kemenes,Kemenes, & Benjamin, 2003; Staras et al., 1999). Such a CGC depolarization is due to an increase in a persistent sodium current. In addition, learning locally down-regulates an axonal potassium current in CGC, leading to a persistent depolarization of its proximal terminals. This plasticity produces a presynaptic facilitation of connections made by CS-activated sensory neurons with the CBIs that drive the CPG and produce the conditioned response (Nikitin, Balaban, & Kemenes, 2013). Finally, the CS–US association decreases intrinsic tonic firing in N3, an inhibitory CPG neuron.
In addition to circuit level analysis, classical conditioning in Lymnaea has enabled investigation of the molecular basis for memory consolidation. Similarly to LTS in Aplysia, associative long-term memory in Lymnaea involves PKA, calmoduline kinase, MAPK, and CREB regulations (Kemenes, Kemenes, Michel, Papp, & Müller, 2006; Michel, Kemenes, Müller, & Kemenes, 2008; Naskar, Wan, & Kemenes, 2014; Ribeiro et al., 2005). In addition, data indicate that a non-coding RNA (Lym-miR-137) is regulated after a single-trial classical conditioning. Using pharmacological approaches and RNA sequencing, it was found that Lym-miR-137 decreases the amount of mRNA for the memory repressor CREB2. After learning, Lym-miR-137 is transiently up-regulated before a down-regulation of CREB2 transcripts occurs. Interestingly, these molecules are both expressed in the CGC that is critically involved in memory formation. This study provided new evidence for the critical role played by non-coding RNAs in memory consolidation (Korneev et al., 2018).
Operant Conditioning of Aerial Breathing in Lymnaea
Lymnaea stagnalis can either use their skin to breathe under water or their lungs to breathe dry air. This bimodal mode of breathing allows animals to adapt their behavior to oxygen availability. In hypoxic water conditions, animals move to the water surface and open their respiratory orifice, the pneumostome. This motivated behavior can be modified by an aversive operant conditioning procedure whereby an aversive tactile stimulation is paired to every spontaneous pneumostome opening. This operant–punisher association elicits an immediate body withdrawal associated with pneumostome closure. As training progresses, both the duration of the behavior and the number of pneumostome openings decrease as compared with “yoked” control animals receiving the same number of punishments but independently of their own operant. Spaced training sessions allow formation of intermediate and long-term memory lasting up to four weeks depending on the inter-session intervals (Lukowiak, Ringseis, Spencer, Wildering, & Syed, 1996; Lukowiak et al., 2003).
The cycles of pneumostome opening/closing movements are generated by a CPG in the central ring ganglia. Three interneurons (RPeD1; IP3; VD4) are interconnected by inhibitory and/or excitatory synapses and are necessary and sufficient to generate the respiratory rhythm (Syed, Bulloch, & Lukowiak, 1990; Syed & Winlow, 1991). RPeD1 is a dopaminergic command-like neuron with synaptic connections to IP3 and VD4, which in turn control pneumostome opening and closing via their synapses with opener/closer motor neurons, respectively. Activation of the aerial respiratory rhythm is induced by hypoxia-related sensory information conveyed from the periphery to the RPeD1.
Operant conditioning decreases genesis of the respiratory rhythm. This effect is associated with a decrease in the ongoing activity of the command-like RPeD1 neuron. Learning also reduces the ability of RPeD1 to trigger patterned bursts of action potentials in IP3 (McComb, Rosenegger, Varshney, Kwok, & Lukowiak, 2005; Spencer, Syed, & Lukowiak, 1999). This change in RPeD1 activity is correlated with an increase in its membrane input resistance and an increase in its intrinsic excitability. Such neuronal plasticity is not observed with “yoked” control procedures. Therefore, the command neuron is a primary locus of the learning-induced plasticity. It is also essential for long-term memory storage, since the surgical ablation of the RPeD1 soma before training, with the neuron’s neurites left intact, impairs long-term memory formation but not intermediate-term memory. In contrast, such an ablation performed one hour after training does not impair long-term memory. Thus, the RPeD1 soma that contains the cell nucleus and genome is required in memory consolidation (Braun & Lukowiak, 2011; Scheibenstock, Krygier, Haque, Syed, & Lukowiak, 2002).
Long-term memory of operant conditioning in Lymnaea involves both DNA transcription and protein synthesis. Recently, inhibiting DNA methylation in animals was found to block the ability of specific environmental or chemical contexts to enhance long-term memory formation (Forest, Sunada, Dodd, & Lukowiak, 2016; Lukowiak et al., 2014; Sunada et al., 2016). As originally described in Aplysia, this finding provides evidence that epigenetic processes, including DNA methylation, regulate long-term memory formation.
Learning and Memory in Limax and Helix
The terrestrial slug Limax maximus can develop remarkably complex associations between odors via short- and long-term aversive classical conditioning, and higher-order classical conditioning. Learning-induced plasticity in odor processing has been investigated in the cerebral ganglion (Gelperin, 1975; Sahley, Gelperin, & Rudy, 1981; for review, see Watanabe, Kirino, & Gelperin, 2008).
Feeding and withdrawal behaviors in the land snails Helix lucorum and Helix pomatia can be modified by habituation, sensitization, or classical conditioning. Synaptic and non-synaptic plasticity, neuromodulation, and molecular processes mediating memory consolidation, reconsolidation, and extinction have been described (for review, see Balaban, 2002). As found in Aplysia, a homologue of vertebrate PKMζ is required for fear conditioning-related long-term memory. It contributes to a synaptic facilitation in the neuronal pathway implicated in the tentacle-withdrawal reflex (Balaban et al., 2015). Identified serotoninergic neurons contribute to the memory reactivation and reconsolidation (Balaban, Vinarskaya, Zuzina, Ierusalimsky, & Malyshev, 2016). Moreover, DNA methylation was found to contribute to the induction of amnesia and memory reconsolidation (Nikitin, Solntseva, Nikitin, & Kozyrev, 2015; Solntseva et al., 2014).
Because of their large neurons and simple nervous systems, gastropod mollusks have provided ideal model organisms for investigating the neurobiological basis of behavioral adaptations and memory formation/maintenance induced by non-associative (habituation, sensitization) and associative (classical and operant conditioning) learning paradigms. Using a variety of reductionist approaches, learning-induced changes in identified neuronal circuits for sensory-motor reflexes, rhythmic, and motivated behaviors, and the molecular cascades in individual neurons that contribute to short (lasting minutes), intermediate (lasting hours), and long-term memories (lasting days) have been analyzed. From these studies, several fundamental concepts have emerged:
1. Learning-induced plasticity involves both synapses and intrinsic membrane properties of neurons at multiple loci in neuronal circuits that are dedicated to sensory processing, decision-making, motor patterning, and/or their interplay.
2. Although a given neuronal circuit, neuron, or synapse can be modified by various learning paradigms, paradigm-specific neuronal plasticity has been identified.
3. Associative learning requires specific molecular association detectors at the single-cell level, such as adenylyl cyclases and/or glutamate receptors.
4. Modulatory transmitters, including monoamines (e.g., serotonin, dopamine) and glutamate, and the resulting regulation of intracellular molecular cascades, contribute to memory formation and maintenance.
5. Short-term neuronal memory relies on covalent changes in pre-existing proteins. In contrast, long-term neuronal memory involves the additional regulation of gene transcription and translation.
Memory maintenance requires molecular switches that transduce transient signals into persistent neuronal engrams.
Gaining fundamental knowledge of invertebrate learning and memory has enabled a bridge between experimental psychology and cellular biology to be established, with a proven applicability to more complex animals. Plasticity of synapses and membrane properties of neurons involved in a given circuit are regarded as the fundamental substrates for learning and memory across all animal phyla. Moreover, monoamines (e.g., 5-HT and dopamine) have been found to play analogous key roles in memory formation in gastropod mollusks and superior vertebrates. Importantly, intracellular signaling pathways, molecules, and the post-transcriptional regulatory mechanisms involved in learning and memory are also shared in gastropod mollusks and vertebrates. Finally, nuclear epigenetic processes such as DNA methylation and histone acetylation have been shown to be involved in long-term memory induction and maintenance in all animals.
Furthermore, it is reasonable to expect, with the ongoing development of new technical, molecular, and genetic tools applied to invertebrates, specifically gastropod mollusks, that new avenues for understanding neuronal operations underlying animal learning and memory will continue to emerge.
We thank Dr. John Simmers for helpful comments and corrections of the manuscript.
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