The seahare a marine snail

Aplysia, a hind-gilled (opistobranch) marine snail (Kandel 1979), is one of the heroes of the cellular revolution in the neurosciences. Its external resemblance to the rabbit earned it the name sea-hare. Yet it is the insides of Aplysia that has turned it into such a highly successful *system in the cellular analysis of simple memory. Quinn (pers. comm.) had defined an ideal *subject for the neurobio-logical analysis of learning as a creature with 10 large neurons, 10 genes, a generation time of 1 week, and the ability to play the cello and recite Shakespeare. Indeed this is not a faithful description of Aplysia, but in the real world, the sea-hare became a useful compromise. Its main assets are a relatively simple nervous system that is readily accessible to experimentation, a simple behavioural repertoire, and a group of capable investigators that have become fascinated by the virtues of the slug.1

The central nervous system of Aplysia is composed of about 20 000 nerve cells arranged in widely spaced ganglia (masses of nerve cells). Some secretory neurons are as big as the entire brain of *Drosophila. Some neurons can be identified from one individual to another by their location, shape, and firing pattern. The system had attracted cellular physiologists (Arvanitaki and Chala-zonitis 1958; Tauc and Gershenfeld 1961; Kandel and Tauc 1965). It was, however, the research on *plasticity and learning that has endowed Aplysia, especially Aplysia californica, with its fame (Kandel and Schwartz 1982; Byrne and Kandel 1996). Following a series of reductive and simplifying steps (*reduction), the cellular and molecular mechanisms of learning in Aplysia have been pursued from the behaving animal, via preparations of isolated ganglia, to identified nerve cells and *synapses in culture (Carew et al. 1971; Rayport and Schacher 1986; Bartsch et al. 1995; Frost et al. 1997; Hawkins et al. 1998). This system is the epitome of the reductionist approach to memory, and as such demonstrates both the advantages and the shortcomings of the approach.

Like all organisms with a nervous system, Aplysia display a repertoire of defensive (e.g. withdrawal) and appetitive (e.g. feeding) reflexes. The analysis of learning in Aplysia has focused mainly on the defensive reflexes (Kandel 1976; Byrne 1985). These can be illustrated by the gill-and-siphon withdrawal reflex (GSWR). The gill is the external respiratory organ of Aplysia. It is housed in the mantle cavity on the dorsal side of the animal. The cavity is a respiratory chamber covered by the mantle shelf. At its posterior end, the shelf forms a fleshy spout, called the siphon. The siphon protrudes out of the mantle cavity between wing-like extensions of the body wall, called parapodia. If a tactile "stimulus is applied to the siphon or mantle shelf, a two-component reflex is elicited. One component is contraction of the siphon and its withdrawal behind the parapodia. The other is contraction of the gill and its withdrawal into the mantle cavity. The GSWR can be "habituated by repetitive monotonous tactile stimuli to the skin; "sensitized by noxious stimuli to the tail or head; and undergo "classical conditioning. This is achieved by pairing a gentle stimulus to the siphon or gill (the conditioned stimulus) with a noxious stimulus to the tail or head (the unconditioned stimulus), so that the conditioned stimulus comes to evoke intense withdrawal (the conditioned response).

In intact Aplysia the GSWR is controlled by both the central and the peripheral nervous systems. Most of the cellular analysis of learning has been performed in the central nervous system, particularly in the abdominal ganglion. This ganglion was found to subserve a substantial portion of the habituation, sensitiza-tion, and classical conditioning of the GSWR. Multiple sites of plasticity have been identified in the abdominal ganglion, but the attention has been focused primarily on one site: the synapse between the sensory neurons and the gill or siphon motor neurons (Kandel and Schwartz 1982; Byrne and Kandel 1996; Figure 5). It has been proposed that part of the behavioural plasticity of the GSWR could be accounted for by use-dependent modifications in this synapse. In brief, the cellular analogue of habituation was portrayed as presynaptic depression, induced by repetitive monotonous firing. As this depression involves only the modified synapse, it is said to be 'homosynaptic'. Sensitization was portrayed as synaptic facilitation, induced in the presynaptic terminal of the aforementioned sensory-to-motor

Heterosynaptic

Fig. 5 A highly simplified scheme of a fragment of the circuit that subserves the gill-withdrawal reflex and its modification by experience in Aplysia. The reflex could be elicited by a tactile stimulus applied to the siphon skin. Repetitive, monotonous tactile stimuli result in habituation of the reflex.A shock to the tail results in sensitization of the reflex. Classical conditioning is obtained by pairing the shock to the tail with a light tactile stimulus to the siphon, so that this tactile stimulus comes to evoke intense withdrawal on subsequent applications in the absence of the shock. Probably hundreds of nerve cells and thousands of synapses subserve the reflex in the intact animal; only a selection of types of cells and synapses are depicted in the scheme. IN, interneuron; MN, motor neuron; SN, sensory neuron. The presynaptic terminal of the sensory-to-motor synapse, denoted by a black triangle (left-hand side), was so far the focus of much of the cellular and molecular analysis of the reflex. *Plasticity of this synapse contributes both to the short- and to the long-term *phases of memory in the reflex. For further details see text.

Fig. 5 A highly simplified scheme of a fragment of the circuit that subserves the gill-withdrawal reflex and its modification by experience in Aplysia. The reflex could be elicited by a tactile stimulus applied to the siphon skin. Repetitive, monotonous tactile stimuli result in habituation of the reflex.A shock to the tail results in sensitization of the reflex. Classical conditioning is obtained by pairing the shock to the tail with a light tactile stimulus to the siphon, so that this tactile stimulus comes to evoke intense withdrawal on subsequent applications in the absence of the shock. Probably hundreds of nerve cells and thousands of synapses subserve the reflex in the intact animal; only a selection of types of cells and synapses are depicted in the scheme. IN, interneuron; MN, motor neuron; SN, sensory neuron. The presynaptic terminal of the sensory-to-motor synapse, denoted by a black triangle (left-hand side), was so far the focus of much of the cellular and molecular analysis of the reflex. *Plasticity of this synapse contributes both to the short- and to the long-term *phases of memory in the reflex. For further details see text.

synapse by "neurotransmitters that are released from interneurons and encode the sensitizing stimulus (Figure 5). As this facilitation involves multiple types of synapses, it is 'heterosynaptic'. Classical conditioning of the GSWR was portrayed as sharing cellular mechanisms with sensitization. It is also activity-dependent presynaptic facilitation; however, in contrast with sen-sitization, which enhances the responsiveness to subsequent stimulation of the skin at any location, the facilitation in classical conditioning is specific to the pathway that has mediated the conditioned input ("coincidence detection). This is hence a pathway-specific, activity-dependent presynaptic facilitation. Multiple molecular mechanisms have been suggested to account for the "acquisition and short-term retention of the synaptic facilitation. They include activation of "intracellular signal transduction cascades by the facilitatory neurotransmitters), phosphorylation (by "protein kinases) of synaptic proteins (e.g. "ion channels), and modulation of transmitter release (Kandel and Schwartz 1982). These simplified cellular "models were later extended, enriched, and modified to include additional synaptic sites and mechanisms (e.g. Byrne and Kandel 1996).

Because of lack of space, we will not concern ourselves here with the fine details of the Aplysia story, but rather with a few generalizations only. The cellular analysis of Aplysia reflexes has shown that a significant component of the circuit that subserves simple learning could be pinned down to the "level of identified neurons and synapses. This analysis was the first to demonstrate the central role of cyclic adenosine monophosphate in memory (Cedar et al. 1972; "CREB), and the multiplicity of time- and "context-dependent mechanisms of plasticity in a single cell. It has also demonstrated that at least part of the loci that subserve short-term memory also subserve long-term memory. Further, analysis of plasticity in the GSWR has provided much support for the "zeitgeist proposal that long-term memory storage relies on modulation of gene expression (Goelet et al. 1986; Martin et al. 1997a,b; "consolidation, "immediate early genes, "protein synthesis). It is noteworthy that in recent years, much of the analysis of learning in Aplysia has practically merged with the cellular biology of "development. This may reflect a genuine homology between learning and development. Yet the focus on molecular and cellular mechanisms, which are shared with other disciplines in the life sciences, may also attest to the current difficulty in switching, even in a "simple system, to the more global level of analysis, which is critical for understanding memory, i.e. that of concerted circuit activity that ultimately encodes "internal representations in the behaving organism.

Over the years, the appreciation of the complexity of the Aplysia system has increased, and the highly simplified models gradually matured into more realistic ones (Glanzman 1995; Byrne and Kandel 1996; Fischer et al. 1997; Bao et al. 1998; Lechner and Byrne 1998; Royer et al. 2000). Attempts are also being made to elucidate the cellular bases of apetitive reflexes (Lechner et al. 2000), as well as of a more complex form of learning, "instrumental conditioning (Nargeot et al. 1999). Aplysia is still our main source of information about the molecular changes that take place in neurons up to a few days after training ("long-term potentiation addresses a shorter time window). This is evident among others from the references made to it in many entries in this book. Admittedly, the memory feats of Aplysia are modest (even the classical conditioning of the GSWR is only of the a type, namely, modification of a pre-existing behaviour and not acquisition of a novel one). But no doubt, without the remarkable work on Aplysia, the molecular and cellular biology of neuronal plasticity, learning, and memory would have been much, much duller. There is still one take home message that is worth mentioning here. The analysis of neuronal plasticity in Aplysia has unveiled an impressive inter-and intra-cellular molecular complexity that keeps growing. This should be noted by orthodox reductionists, who erroneously think that reducing a system implies simplifying it. The opposite might be the case.

Selected associations: CREB, Reduction, Simple system, Synapse

'The major driving force behind the Aplysia project, Eric Kandel, shared the 2000 Nobel prize for Medicine.

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