Larval zebrafish display dynamic learning of aversive stimuli in a constant visual surrounding

Balancing exploration and anti-predation are fundamental to the fitness and survival of all animal species from early life stages. How these basic survival instincts drive learning remains poorly understood. Here, using a light/dark preference paradigm with well-controlled luminance history and constant visual surrounding in larval zebrafish, we analyzed intra- and intertrial dynamics for two behavioral components, dark avoidance and center avoidance. We uncover that larval zebrafish display short-term learning of dark avoidance with initial sensitization followed by habituation; they also exhibit long-term learning that is sensitive to trial interval length. We further show that such stereotyped learning patterns is stimulus-specific, as they are not observed for center avoidance. Finally, we demonstrate at individual levels that long-term learning is under homeostatic control. Together, our work has established a novel paradigm to understand learning, uncovered sequential sensitization and habituation, and demonstrated stimulus specificity, individuality, as well as dynamicity in learning.

Learning while being exposed to a stimulus (i.e., nonassociative learning) is of great importance in that it triggers intrinsic constructs for subsequent recognition of that stimulus and provides a foundation for associative learning (e.g., learning about relations between stimuli in Pavlovian conditioning and stimuli responses-outcomes in instrumental conditioning). Nonassociative learning precedes associative learning in the evolutionary sequence and involves a broad range of behavioral phenomena including habituation, sensitization, perceptual learning, priming, and recognition memory (Pereira and van der Kooy 2013; Ioannou and Anastassiou-Hadjicharalambous 2018).As the simplest learning form, habituation is defined as the progressively reduced ability of a stimulus to elicit a behavioral response over time (Glanzman 2009; Rankin et al. 2009; Thompson 2009). Such a response reduction is distinguished from sensory adaptation and motor fatigue and is often considered adaptive in that it helps animals to filter out harmless and irrelevant stimuli (Rankin et al. 2009). Since an early study of EEG arousal in cats (Sharpless and Jasper 1956), the habituation phenomenon has been widely documented in invertebrates such as C. elegans (Rankin and Broster 1992; Rose and Rankin 2001; Giles and Rankin 2009; Ardiel et al. 2016) and Aplysia (Glanzman 2009) as well as in vertebrates such as rodents (Bolivar 2009; Salomons et al. 2010; Arbuckle et al. 2015), zebrafish (Best et al. 2008; Wolman et al. 2011; Roberts et al. 2016; Randlett et al. 2019; Pantoja et al. 2020) and humans (Bornstein et al. 1988; Coppola et al. 2013).Accompanying habituation is a process termed sensitization, which in contrast enhances responses to a stimulus over time (Kalivas and Stewart 1991; McSweeney and Murphy 2009; Robinson and Becker 1986). This counterpart of habituation may also be adaptive if it helps animals avoid potentially risky or costly situations (Blumstein 2016; King et al. 2007). Like habituation, sensitization has also been documented in a phylogenetically diverse set of organisms (Cai et al. 2012; Kirshenbaum et al. 2019; Tran and Gerlai 2014; Watkins et al. 2010), suggesting an evolutionarily conserved biological underpinning for both processes. Furthermore, these simple learning forms are observed in various functional outputs of nervous systems ranging from simple reflexes (Blanch et al. 2014; Pantoja et al. 2020; Pinsker et al. 1970; Randlett et al. 2019) to complex cognitive phenotypes (Bolivar 2009; Leussis and Bolivar 2006; Thompson and Spencer 1966) and may represent deeper neurobiological constructs associated with anxiety-memory interplay (Morgan and LeDoux 1995; Ruehle et al. 2012; Sullivan and Gratton 2002). Therefore, understanding basic building blocks of habituation and sensitization is essential to fully understand complex behaviors.Habituation and sensitization have been reported with short-term (intrasession) and long-term (intersession) mechanisms in a number of systems (Rankin et al. 2009; Thompson 2009). Short-term mechanisms sensitize or habituate a response within a session (Meincke et al. 2004; Leussis and Bolivar 2006; Rahn et al. 2013; Byrne and Hawkins 2015). In contrast, long-term mechanisms retain memory formed in previous session and use it to modify behavioral responses in a subsequent session (Rankin et al. 2009).Although both short- and long-term learning and memory have been demonstrated in young larval zebrafish (Wolman et al. 2011; O''Neale et al. 2014; Roberts et al. 2016; Randlett et al. 2019), so far, most paradigms use unnatural stimuli and are designed without integrating sensitization and habituation in the same paradigm. The latter limitation is crucial as the influential dual-process theory, proposed by Groves and Thompson (1970), suggests that the two learning forms interact to yield final behavioral outcomes and therefore assessment of only one process might be confounded by alteration in the other process (Meincke et al. 2004).In this study, we examined stimulus learning in a large population of larval zebrafish using a light/dark preference paradigm over four trials across 2 d. Light/dark preference as a motivated behavior is observed across the animal kingdom (Serra et al. 1999; Bourin and Hascoët 2003; Gong et al. 2010; Lau et al. 2011). Larval zebrafish display distinct motor behaviors that are sensitive to the intensity of both preadapted and current photic stimuli (Burgess and Granato 2007; Burgess et al. 2010; Facciol et al. 2019). In our paradigm with well-controlled luminance history and constant visual surrounding, larval zebrafish generally display dark avoidance and center avoidance (also known as thigmotaxis) with heritable individual variability and are considered fear- or anxiety-related (Steenbergen et al. 2011; Schnörr et al. 2012; Bai et al. 2016; Wagle et al. 2017; Dahlén et al. 2019). From an ethological perspective, the extent of avoidance is likely a readout of the circuitry that balances anti-predation (i.e., avoid the dark and the center) and free exploration (i.e., approach the dark and the center). As described below, we have uncovered stimulus-specific temporal dynamicity of learning (both short term and long term), as well as individual differences in learning that are under homeostatic control.