Ontogeny of classical and operant learning behaviors in zebrafish

Abstract

The performance of developing zebrafish in both classical and operant conditioning assays was tested with a particular focus on the emergence of these learning behaviors during development. Strategically positioned visual cues paired with electro-shocks were used in two fully automated assays to investigate both learning paradigms. These allow the evaluation of the behavioral performance of zebrafish continuously throughout development, from larva to adult. We found that learning improves throughout development, starts reliably around week 3, and reaches adult performance levels at week 6. Adult fish quickly learned to perform perfectly, and the expression of the learned behavior is manifestly controlled by vision. The memory is behaviorally expressed in adults for at least 6 h and retrievable for at least 12 h. [Supplemental material is available for this article.] The ability to associate two environmental cues, as in classical conditioning, or to correlate one's behavior with its consequences , as in operant conditioning, are processes often essential for an animal's survival (Skinner 1984). While there are studies that have followed the ontogeny of learning behaviors in animal model systems of classical conditioning paradigms (Campbell and Ampuero 1985; Moye and Rudy 1985; Paczkowski et al. 1999; Raineki et al. 2009), the ontogeny of operant learning, in particular , remains much less explored. The ability for classical conditioning is pervasive across the animal kingdom and often interpreted as the most basic and robust form of associative learning; however, it has been shown that adding an operant component to a learning task may increase performance, or show it where it was not present before (Heisen-berg et al. 2001; Moore 2004). Zebrafish is an exceptional model of vertebrate neural development and amenable both to forward genetics and genetic manipulation (Neuhauss 2003). The transparent larvae are uniquely suited to functional imaging and have recently served to uncover fundamental mechanisms in fish locomotor control (Orger et al. 2008; Portugues and Engert 2009). Behavior is the ultimate functional readout of neural activity; indeed, behavior is the ultimate evolutionary reason for the existence of brains and the changes in behavior during development should reflect the changes the nervous system undergoes as the larval organism develops into the adult. While there are well-established learning paradigms for Drosophila and rodent experimental models, zebra-fish still lacks a systematic learning characterization. We describe here an approach to implement and test such a learning assay that utilizes the fact that zebrafish can see and track visual stimuli and clearly perform visually evoked locomotor behaviors as early as 5 d post-fertilization (dpf) (Easter and Nicola 1996; Budick and O'Malley 2000). To that end we use visually guided stimuli combined with a noxious electroshock to control learning behaviors. We characterize these assays and specifically obtain a full ontoge-ny of operant learning fr...

Zebrafish forebrain and temporal conditioning

“…Because adult zebrafish are not suitable for non-invasive whole-brain calcium imaging (other than imaging the dorsal surface of the brain after removal of the skull as shown by Aoki et al [9]), a recent study examined the time course of conditioned learning in larval zebrafish [66]. The fish were trained in an aversive visuospatial classical conditioning task in a daily session (six trials per session) continuously from 7 to 42 dpf.…”

Zebrafish learn to forage in the dark

“…The ability to perform associative learning increases with age. In experiments that used swimming in response to visual cues, Valente et al, (2012) found larvae younger than 4 weeks old to be unresponsive to classical conditioning, but to respond to operant conditioning by electric shocks as young as 3 weeks old. Our results suggest that larvae may begin to learn by 15 dpf when offered the reward of prey.…”

“…From an early age, zebrafish are also responsive to operant conditioning. For example, larvae can be trained to orient their swimming to visual cues by negative reinforcement with an electric shock, and they improve in this task with age to the extent that larvae perform almost flawlessly by 4 weeks of age (Valente et al, 2012). The present study tested whether improvement in foraging in the dark is a consequence of associative learning by measuring the foraging of fish that were experimentally restricted from exposure to the flow of prey.…”

Spontaneous alternation behavior in larval zebrafish

“…Adult zebrafish perform well in multiple memory-related experiments including olfactory conditioning (Braubach et al, 2009), spatial alternation (Cognato et al, 2012), and associative (Sison and Gerlai, 2010;Valente et al, 2012) and non-associative (Tran and Gerlai, 2014) learning. Mnestic studies using larval zebrafish, in contrast, have only commenced recently (Roberts et al, 2013).…”