Background

Associative learning enables animals to prepare for behaviorally relevant events, thus being highly conserved. Learning to predict events in the environment and which stimuli tend to occur together help us interact effectively with our environment. This cognitive process has been shown to play a role in a wide diversity of behaviors, including interactions with predators, prey, rivals, and mates. Thus, learning is expected to be essential for survival and reproduction in many species. The study of such associative learning in simple model systems, like the fruit fly Drosophila melanogaster, facilitates its understanding especially at multiple levels of analysis. This model allowed researchers to uncover principles and mechanisms of learning and memory as it offers a wide range of powerful genetic tools to dissect intrinsic molecular mechanisms (McGuire et al., 2005; Pitman et al., 2009).

During Pavlovian classical conditioning, animals learn to associate a salient unconditioned stimulus with a neutral stimulus. Olfactory classical conditioning is a well-studied form of associative learning in the fruit fly D. melanogaster (see Busto et al., 2010). Subsequently, other methods to measure various types of learning and memory have been proposed, including courtship conditioning (Koemans et al., 2017), aversive phototaxic suppression assay (Ali et al., 2011), and wasp-exposure conditioning (Kacsoh et al., 2019).

Regarding visual learning, it has been extensively studied using the so-called flight simulator (Wolf and Heisenberg, 1991). Here, a tethered fly is flying stationarily in the center of a cylindrical virtual arena panorama where different visual stimuli are displayed. Combined with heat punishment, the setup changes into an operant conditioning paradigm in which the fly learns to control the appearance of the punisher. Like this, animals have to learn from the consequences of their own and voluntary actions in order to avoid the punishment. Other studies show that populations of flies are able to associate the color of the illumination alone (Vogt et al., 2015) or in combination with odors (Vogt et al., 2014; Thiagarajan et al., 2022; Okray et al., 2023) with an appetitive or aversive stimulus.

Despite the relative abundance of aversive learning assays in which flies operantly avoid heat by choosing certain orientations relative to landmarks, only a few behavioral paradigms for appetitive visual learning in adult Drosophila have been reported to date (Heisenberg, 1989; Schnaitmann et al., 2010; Vogt et al., 2014). Here, we established a new behavioral paradigm for visual classical conditioning in freely-walking adult Drosophila. In their natural environment, fruit flies primarily move by walking rather than flying. Studying them in a walking state allows us to better understand their visual learning abilities in a context that aligns with their ecological niche. This assay allows analysis of the behavior of individual flies while they make a choice, learn, and memorize (short-term memory) visually rewarded associations. The paradigm described here is based on a simple behavioral assay to study orientation behavior in flies (Buridan paradigm), which can be easily assembled with a few available supplies (Figure 1). Particularly, this visual learning is based on the spontaneous numerical preference of flies, which manifests as a larger occupancy next to the higher numerosity in a two-choice test (Bengochea et al., 2023). The setup consists of presenting two visual sets of different numbers of objects that are located opposite to each other, in order to be processed individually and not treated as a whole. Since flies have a spontaneous preference for larger sets of objects, the appetitive reward is placed next to the non-preferred numerical set (lower numerosity), while the innately preferred one is water-paired (Figure 1B). A single fly is placed on the platform (Figure 1C). During the 3-min training trial, the fly explores the arena and eventually reaches the sugar and water. Two hours after training, the learned preference of the fly is evaluated during a 5-min testing phase (Graphical overview). By analyzing the walking occupancy in the arena, we are able to observe a decrease or change in their natural tendency to prefer the larger quantities due to the association of the sugar with the visual set. The representative results of this assay reveal that the application of the protocol impacts on the fly’s numerical preference (Figure 2, Bengochea et al., 2023). A single training trial of 3 min is sufficient to teach flies to associate an appetitive reward with a visual set that lasts at least two hours and to partially or totally reverse a spontaneous tendency.