Background

Habituation is a fundamental form of learning that is conserved across species. In habituation, the response to a persistent inconsequential stimulus is reduced, leading to an enhanced ability to attend to more pertinent stimuli. A defining feature of the habituated state is the presence of a latent ability to respond to the familiar stimulus (Thompson and Spencer, 1966; Rankin et al., 2009; Ramaswami, 2014). This latent ability to respond is made evident by the effect of a novel external stimulus, which results in override of habituation, i.e., restoration of a robust response in the habituated animal. This can also be observed when an animal is motivated to attend to the familiar stimulus (Kato et al., 2015). The neural mechanism of habituation has been extensively studied across different species and different modalities, particularly in Aplysia, Drosophila, and mouse, using a variety of different behavioral protocols (Castellucci et al., 1970; Krasne and Edwards, 2002; Engel and Wu, 2009; Ogg et al., 2015). In Aplysia, both homosynaptic depression at the sensory-motor synapse (Castellucci et al., 1970) and potentiation of an inhibitory interneuron synapse (Bristol and Carew, 2005) have been suggested as underlying mechanisms of habituation of the gill-induced siphon withdrawal reflex. Later studies in Drosophila and vertebrates indicate that heterosynaptic potentiation of inhibition can lead to olfactory, auditory, or gustatory habituation (Das et al., 2011; Paranjpe et al., 2012; Ramaswami, 2014; Kato et al., 2015). However, the neural correlates of habituation override in these types of habituation are only just beginning to be investigated (Trisal et al., 2022). Here, we first describe the protocol for the proboscis extension reflex (PER), which has been used previously to study gustatory habituation in Drosophila (Duerr and Quinn, 1982; Le Bourg, 1983; Fois et al., 1991; Engel and Wu, 2009; Paranjpe et al., 2012), and then a protocol to induce override of PER habituation following exposure to a novel sensory stimulus (Paranjpe et al., 2012; Trisal et al., 2022). The novel stimulus used is a non-invasive gustatory stimulus (10% yeast), which the flies have not encountered before, that is presented to the proboscis. Compared to previous publications on PER (Shiraiwa and Carlson, 2007; Li et al., 2022), we provide a simpler and potentially less traumatic method to immobilize the flies. In addition, our protocol (also used by Paranjpe et al., 2012) is unique in that it exposes the flies continuously to 10% sucrose (while preventing ingestion at the same time) to induce more robust PER habituation than in the original studies. Our approach allows a more dynamic range and time to study habituation and dishabituation. While our method requires approximately 15 min for analysis of an individual fly, it requires far fewer flies to reach statistical significance.