NATURE NEUROSCIENCE VOLUME 8 | NUMBER 12 | DECEMBER 2005 1639

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Nematodes learn: now what?William G Quinn

A study in Nature reports that nematodes can learn to associate different chemosensory stimuli with illness and to avoid these stimuli in a choice test. Elevated serotonin in a particular type of neuron was critical for this learning.

The nematode worm Caenorhabditis elegans has been an important model for four decades now, because of its rapid genetics, simple ner-vous system (302 neurons) and stereotyped development. What the worm seemed to lack was sophisticated behavior—careers have been built and Nobel prizes gained study-ing genetically induced variants in swim-ming, egg-laying and death. Now Zhang et al.1, building on earlier work2,3, persuasively report in Nature that nematodes can learn to associate different chemosensory stimuli with illness, and to avoid these stimuli in a choice test. The learning is of an unusual type, dis-covered 60 years ago in rats.

In the mid 1950s, John Garcia and his col-leagues discovered that rats made ill (nause-ated) by radiation or toxins avoided foods that they had tasted some time earlier4. This induced behavioral change differed from clas-sical conditioning in several ways. The effect was strikingly strong and long lasting, the pairing was confined to tastes and nausea and, particularly, it lacked the requirement for close temporal association of stimulus and reinforce-ment—the taste could occur some hours before the induced nausea, rather than the interval of a second or so that is typical of classical condi-tioning or electric shock–reinforced learning. Garcia’s work was controversial for years but is now established as authentic learning, with useful applications in ecology and patient care5. The effect has been demonstrated in mammals, squid and garden slugs. The worms of Zhang et al. also seem to learn in this way.

Nematodes feed on bacteria. Some strains of bacteria are pathogenic; worms that feed on them will eventually die. Worms raised for 4 hours on pathogenic bacteria will later avoid them when given a choice. Associative learning by definition depends on temporal pairing between stimulus and reinforcement. In the case of taste (or odor) aversion learn-ing, the requirement for pairing is loosened

The author is in the Department of Brain and

Cognitive Sciences, 46-5009, Massachusetts

Institute of Technology, 77 Massachusetts

Avenue, Cambridge, Massachusetts 02139, USA.

e-mail: cquinn@mit.edu

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Starved for 4 hoursExposed to PA14 for 4 hours

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Figure 1 Discriminative learning in C. elegans. (a) Training and testing scheme, part 1. A sample of worms was raised on pathogenic P. aeruginosa (PA14) bacteria for 4 hours, then transferred for a further 4 hours to non-pathogenic bacteria of a different species (S. marcescens), which presumably have a different smell and taste. The worms were then transferred to a test plate, placed between samples of the two bacterial species (both pathogenic). After 1–2 hours, they were immobilized with sodium azide, and the worms on each bacterial sample were counted. (b) Training and testing, part 2. A new sample of worms were trained and tested as above, but with the pathogenicity switched to the other species (pathogenic and non-pathogenic strains of each species were, as far as possible, coisogenic). A relative shift in choice bias from S. marcescens in a to P. aeruginosa in b indicates discriminative (associative) learning to diffusible cues. (In the actual experiments, two more samples of worms were trained and tested exactly as described above, but with the order of exposure to species switched during training—S. marcescens first.) (c) Altered choice bias during testing. Animals exposed to pathogenic PA14 and harmless S. marcescens avoid PA14 more than animals exposed to harmless P. aeruginosa and pathogenic S. marcescens. (d) Data from c, replotted to show raw avoidance of one of the species (P. aeruginosa). Panels c and d reproduced from ref. 1 with permission from the authors. ***P < 0.001, *P < 0.05, n ≥ 4 assays. Error bars represent s.e.m.

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from seconds to hours, making pairing studies less conclusive. Given this limitation, Zhang et al. did the necessary controls for associative learning as well as possible, using a discrimi-native learning assay with built-in controls (Fig. 1a,b). They used pathogenic and non-pathogenic variants of two species of bacteria (Pseudomonas aeruginosa and Serratia marc-escens). Four groups of worms were tested in four different experiments. Each experiment involved placing the worms for 4 hours on pathogenic bacteria of one species and then for 4 hours on non-pathogenic bacteria of the other species, concluding with a choice test between species. In different experiments, the pathogenicity was switched from one species to the other, and the order of species presenta-tions was reversed. When choice percentages were averaged over all the experiments, there was a significant overall tendency of the worms to avoid the bacterial odor (or taste) experi-enced with pathogenicity (Fig 1c). Switching pathogenicity from one bacterial species to the other during training altered the worms’ later choice percentage between bacteria by about 40% during testing, averaged over all the experiments. Discriminative conditioning assays of exactly this type have been success-fully used to condition bees, fruit flies, slugs and Aplysia vaccaria, among other species6. Now nematodes can be trained in this way.

Zhang et al. then went on to use available mutants and transgenic worms to identify molecular components of the learning path-way and to start to define the neuronal circuit involved. Worms with a mutation3 in the gene for tryptophan hydroxylase (necessary to syn-thesize the neurotransmitter serotonin) failed to learn, although they showed normal chemo-sensory choices in non- learning tests. Indeed, raising nematodes on pathogenic bacteria

dramatically increased their serotonin levels in several neurons, including in chemosensory neurons denoted ADF (Fig. 2). Expression of tryptophan hydroxylase cDNA in this neuron partially rescued the learning defect, whereas expressing the cDNA in another neuron, NSM, did not. This suggests (but does not prove) that elevated serotonin in the ADF cells, at least in part, signals the existence of the negative reinforcement—malaise from ingesting pathogenic bacteria.

Nematodes have genes for several serotonin receptors, with mutants available for some of them. One such gene, mod-1, encodes a sero-tonin-gated chloride channel that is known to function in another response to food7. Mod-1 mutants, lacking the receptor, are deficient in the aversive component of the learning behav-ior. Mod-1 is expressed in five interneurons in the worm chemosensory circuit. ADF syn-apses directly onto two of them, AIZ and per-haps AIY, giving a suggestive minimal circuit for aversive learning of three cells—not too shabby. However, most of the mutant effects and the rescues were only partial, and some of the cellular localizations were also incomplete. Moreover, transmitters including serotonin can act at a distance (like hormones), as well as directly across synapses. Laser ablation studies may help refine the circuit further, provided the learning assay can be made strong enough to score small numbers of worms.

C. elegans is an elegant model organism, but is it a model mostly of itself? Certainly its genes, molecules and metabolic pro-cesses are quite similar to ours—the pathway for programmed cell death, present in all animals, was worked out largely from genetic studies in nematodes. In this regard, the implication of serotonin as a reinforcement signal is consistent particularly with work in

Aplysia vaccaria8. What is surprising from the worm work is the finding that serotonin acts through an ion channel–linked receptor, rather than a G protein–coupled one, together with the absence of any evidence for one of the second-messenger systems implicated in learning in so many other species. Given that second- messenger systems are a handy way to produce moderately long- lasting changes in cells, it will be interesting to see from future studies how C. elegans encodes its memories. In particular, it will be revealing to see how long the worms can remember the chemosensory discrimination they have learned.

Another open question also promises new mechanisms: how does C. elegans handle stimulus pairing? Associative learning requires that the stimuli—a bacterial taste or smell and nausea in this case—converge synergistically on some molecular entity to initiate a lasting change in the relevant neurons. In vertebrates, this convergence occurs in most cases onto the voltage-and-ligand-gated NMDA type of gluta-mate receptor. In A. vaccaria and in Drosophila melanogaster, signals from stimuli converge primarily onto the calcium-activated and G protein–activated (type I) adenylyl cyclase enzyme8,9. In nematodes, serotonin seems to be the signal for reinforcement, but it must somehow produce lasting changes in the avoid-ance circuit only for the paired chemosensory stimulus (for example, from P. aeruginosa) but not the unpaired control (for example, from S. marcescens). It is not clear yet how the mod-1 receptor could do this without hidden proper-ties or new molecular partners.

The study by Zhang et al. reflects a real advance in worm sophistication, as well as in human sophistication in understanding them. Given the small nervous system of the worm and the powerful genetic, molecular and ana-tomical tools available to study it, it represents a foot in a very big door.

1. Zhang, Y., Lu, H. & Bargmann, C.I. Nature 438, 179–184 (2005).

2. Bernhard, N. & van der Kooy, D. Learn. Mem. 7, 199–212 (2000).

3. Sze, J.Y., Victor, M., Loer, C., Shi, Y. & Ruvkun, G. Nature 403, 560–564 (2000).

4. Garcia, J., Kimeldorf, D.J. & Koelling, R.A. Science 122, 157–158 (1955).

5. Rozin, P. & Kalat, J.W. Psychol. Rev. 78, 459–486 (1971).

6. Carew, T.J. & Sahley, C.L. Annu. Rev. Neurosci. 9, 435–487 (1986).

7. Ranganathan, R., Cannon, S.C. & Horvitz, H.R. Nature 408, 470–475 (2000).

8. Kandel, E.R. Science 294, 1030–1038 (2001).9. Livingstone, M.S., Sziber, P.P. & Quinn, W.G. Cell 37,

205–215 (1984).

Figure 2 Pathogenic bacteria elevate ADF neuron serotonin levels. (a,b) Serotonin immunoreactivity in wild-type animals fed either Escherichia coli (a) or E. coli and pathogenic P. aeruginosa (b). Note the increase in immunoreactivity (marked by fluorescence) in b, particularly in chemosensory neurons ADF. Figure reproduced from ref. 1 with permission.

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