The struggle to define “learning” and “memory” is a fundamental problem addressed by a multitude of behavioural and neuroanatomy studies. With the relative neural complexity of popular model organisms (135 000 neurons in Drosophila melanogaster, 4 000 000 neurons in mice), it remains a challenge to understand learning on anything more than the level of neuronal wiring and regional firing, much less to isolate the biochemical representation of a memory.

The nematode Caenorhabditis elegans, despite having only 302 neurons, can undergo classical (Pavlovian) conditioning, and can therefore be tested in an associative learning paradigm. When worms are given an odourant (such as benzaldehyde, Bnz) during a period of starvation, their natural preference for this smell switches from attractive to aversive [1]. In this way, worms are able to integrate internal and external cues in order to behaviourally adapt to their environment. This presents a simple model organism with which to study the molecular mechanisms of learning, something that is neither well understood nor easily characterized in a more complex animal.

Classical conditioning is defined as the association of a conditioned stimulus (CS) and an unconditioned stimulus (US) to produce a conditioned response. In the Bnz-starvation paradigm, the US is the innately aversive physiological state of starvation. The CS is Bnz, which untrained worms find naively attractive. Worms can be trained by exposure to the CS in the presence of the US, represented in the lab by the presence of Bnz in absence of food. Learning occurs when an association between Bnz and starvation is formed such that worms actively avoid a point source of the Bnz CS. Since this learning paradigm involves starvation, it is clear that the presence or absence of food represents an important external signal to worms. This regulation of “satiety-state” in worms has been shown to be mediated by serotonergic signaling [2,3].

Serotonin is a biogenic amine neurotransmitter that mediates multiple food-related processes in worms. The effects of applying exogenous serotonin to worms were first characterized in Horvitz et al. (1982), which described three main behavioural responses: depressed locomotion, stimulated pharyngeal pumping, and increased rate of egg laying [4]. These phenomena correlated with those observed in earlier studies describing food-mediated behavioural responses in worms [5]. Furthermore, later studies clarified the causal role of endogenous serotonin signaling in these phenomena. The modulation of the satiety state was linked to serotonin by showing that exogenous serotonin reversed starvation-responsive behaviour [6]. Likewise, the “depressed locomotion” was a result of the role of endogenous serotonin signaling in mediating an “enhanced slowing” response of fasting worms [2]. Increased pharyngeal pumping was found to also result from the presence of food in a worm’s environment, and multiple serotonin-binding receptors were implicated as necessary for modulating this response [7]. In addition, increased rate of egg-laying was also found to be mediated by serotonin-binding to different serotonin receptors [7].

These data implicate serotonergic signaling as a key regulator of satiety status in worms. Serotonin signaling has also been implicated in the aforementioned starvation-odourant associative learning paradigm. This was accomplished by demonstrating that the presence of food during the training period could block the Bnz-starvation association, and that this phenomenon could be recapitulated with the
application of exogenous serotonin. These two training paradigms described a “food block” and a “serotonin block”, respectively [3]. This research also showed that worms deficient in serotonin signaling (mutant for the genes tph-1 or cat-4, involved in serotonin synthesis) were not able to be food blocked, and learned to associate Bnz with starvation in the presence of food. This raises the question of whether serotonin signaling is not just important as a satiety signal, but whether the lack of serotonin signaling is the nature of the starvation US. Therefore, previous research indicates that the presence or absence of serotonin signaling appears to mediate the US arm of the associative learning paradigm by regulating a worm’s satiety state.

C. elegans has five canonical serotonin receptors, and four receptors with strong serotonin receptor homology. Of the canonical receptors, four (ser-1, ser-4, ser-5, and ser-7) are G-protein coupled receptors and one (mod-1) is a ligand-gated ion channel [7]. Different receptors are involved in mediating different aspects of the food response, including pharyngeal pumping, locomotion, and increased
egg-laying [8-12].

This study shows that worms mutant for the five canonical serotonin receptors (“quintuple mutants”) are defective in their ability to be serotonin blocked. The receptors necessary for this phenomenon were investigated, and it was found that the loss of ser-4 alone replicated the minimal serotonin blocking seen in quintuple mutants, implicating the SER-4 receptor in mediating the US arm of the Bnz-starvation classical conditioning paradigm.

The goal of this study is to explore the mechanisms of Bnzstarvation associative learning in C. elegans in order to better understand the nature of memory formation and storage on a molecular level. Worms are a good model organism with which to investigate these pathways because of their structural simplicity and facile genetics. In addition, many serotonin receptors in worms are homologues of human serotonin receptors [7]. The mechanisms involved in their associative learning may comprise a simplification of the mechanisms involved in mammalian associative learning. Based on the prior research, this study hypothesizes that serotonin signaling is involved in mediating the starvation/ food signal in C. elegans, which, upon integration with Bnz, results in associative learning of the Bnz-starvation association.