Research report
Latent learning in zebrafish (Danio rerio)

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Abstract

The zebrafish may represent an excellent compromise between system complexity and practical simplicity for behavioral brain research. It may be particularly appropriate for large scale screening studies whose aim is to identify mutants with altered phenotypes or novel compounds with particular efficacy. For example, the zebrafish may have utility in the analysis of the biological mechanisms of learning and memory. Although learning and memory have been extensively studied and hundreds of underlying molecular mechanisms have been identified, this number may represent only the fraction of genes involved in these complex brain functions. Thus large scale mutagenesis screens may have utility. In order for such screens to succeed, appropriate screening paradigms must be developed. The first step in this research is the characterization of learning and memory capabilities of zebrafish and the development of automatable tasks. Here we show that zebrafish is capable of latent learning, i.e. can acquire memory of their environment after being allowed to explore it. For example, we found experimental zebrafish that experienced an open left tunnel or an open right tunnel of a maze during the unrewarded exploration phase of the test to show the appropriate side bias during a probe trial when they had to swim to a group of conspecifics (the reward). Given that exploration of the maze does not require the presence of the experimenter and the probe trial, during which the subjects are video-recorded and their memory is tested, is short, we argue that the paradigm has utility in high-throughput screening.

Introduction

The zebrafish has been one of the favored model organisms of embryology [48] and, due to the numerous and extensive developmental studies using genetic approaches conducted with this species, by now zebrafish is one of the most frequently employed animal model organisms of genetics too [27]. As a result of the accumulated genetic knowledge and the large number of genetics tools developed for this species, the zebrafish has been gaining popularity in behavioral brain research as well [42]. Research using genetic methods has been successful in the analysis of the biological mechanisms of brain function [13]. Thus, zebrafish with its strong genetics toolset may also have utility in behavioral brain research in addition to the classical laboratory rodents, mice and rats, and to the lower order organisms such as the nematode or the fruit fly.

Furthermore, when comparing these evolutionarily highly diverse laboratory study species, the zebrafish appears to enjoy some advantages. Although it is a vertebrate that possesses a complex brain whose major anatomical layout [47], [38] and neurochemistry [12], [22], [34], [35] are similar to those of mammals including our own species, it is almost as easy to keep in the laboratory and is also almost as prolific as non-vertebrate laboratory organisms. Thus, the zebrafish appears to strike an optimal compromise between system complexity (brain function in this case) and practical simplicity (pragmatic considerations including cost). Particularly useful this species may be when one considers that a small zebrafish rack (150 cm tall, 120 cm wide, 30 cm deep) can house as much as 4000 fully mature adult zebrafish. This feat is accomplishable due to the small size (maximum length is 4 cm) and social nature (shoaling) of zebrafish. Shoaling is a typical aggregation behavior whereby conspecifics stay close to each other, usually within an inter-individual distance of 3–4 body lengths, and form a group (e.g. [32], [33]). If one also considers that mutagenesis methods, including chemical (ethyl-nitroso-urea, ENU) [29], viral vector mediated (insertional) [2], and other (e.g. transposon induced gene breaking) mutagenesis techniques [44] have been worked out for the zebrafish, it becomes clear that this species is an excellent choice for forward genetics [10], [3].

But what is the merit of conducting random mutagenesis with the need to screen thousands of mutants for their phenotypical alterations in learning and memory when we already know a couple of hundreds of molecular players [45] involved in these processes? Perhaps instead, one should conduct a thorough analysis of the already known molecular players using, for example, reverse genetics. Indeed, numerous laboratories have taken this latter route and seminal studies have been published on the functional characterization of what is believed to be the key players of learning and memory (e.g. Ref. [26]). However, according to some estimates as many as 40–50% of all genes in the genome of vertebrate species are expressed in the brain of these species [15], which represents a staggering number (about 12–15 thousand genes) compared to the couple of hundred genes with already proven roles in learning and memory. A systematic analysis of the potential involvement of the rest of the genome is thus of high relevance. Discovery of potentially large number of genes involved in learning and memory may be possible using large scale forward genetic screens, but only if proper screening tools, i.e. behavioral testing paradigms are available [25]. The goal of the current paper is to advance our knowledge in this direction by testing zebrafish in a learning task that is easy to administer and one which may be employed for large scale screening.

Although the zebrafish is relatively new in the study of learning and memory as compared to other classical laboratory model organisms [43], by now several studies have shown that this small vertebrate is also capable of performing well in a range of learning tasks. For example, zebrafish showed acquisition of a one trial avoidance learning task [6], performed well in olfactory conditioning [11], shuttle box active appetitive conditioning [36], place conditioning [17], appetitive choice discrimination [5], active avoidance conditioning [51], alternation memory task [50], and most recently in a plus-maze non-spatial and spatial associative learning task [1], [42]. While these results clearly demonstrate the cognitive and mnemonic capabilities of zebrafish, the tasks have not been used in high-throughput screening because they often required extensive and labor intensive training. The possibility of automation, and thus high throughput, however, also appears to be within reach for zebrafish as demonstrated by two recent studies [28], [36]. In the current paper, we introduce a simple learning task based upon spatial exploration, which we argue may also be made high throughput. In this task zebrafish are repeatedly exposed to a maze (Fig. 1) with particular tunnels (left side, right side or both sides) open. This training phase does not require monitoring of behavioral activity and thus can be performed without the presence of the experimenter. The training phase is followed by a short probe trial during which the swim tunnel choice (spatial bias) of the fish is quantified. We call this task a “latent learning” paradigm [8], [46] because the training phase of the test involves no experimenter controlled delivery of reinforcers. Here, we show that zebrafish develop a significant spatial bias in this task and argue that the paradigm will be appropriate for high-throughput screening.

Section snippets

Animals and housing

Wild-type short-fin zebrafish were obtained from a local pet store (Big Al's Aquarium Services Inc., Mississauga, ON, Canada) and were bred in the Vivarium (University of Toronto Mississauga). Subjects used in the current study were from the first filial generation raised and housed in the same vivarium room under identical conditions. The experimental fish were housed (5 fish per tank) in 3 l transparent acrylic tanks (a trapezoid tank with a bottom measuring 22 cm × 9 cm, top measuring 26 cm × 9 cm

Results

Zebrafish are social fish that swim in shoals in nature [19] as well as in captivity [41], [32], [33]. The fact that we ran 10 fish at a time during each training trial, made the novel test environment less aversive and allowed the fish to properly habituate to this environment. As a result, experimental fish actively explored the maze throughout training and signs of fear, e.g. freezing, erratic movement or jumping, [4], [37], [23] were not observed. Similarly, fear responses were absent

Discussion

The question this study attempted to answer was whether fish exposed to the maze without any reward could learn about the maze and could behave differently as a result of acquiring memory of the maze in a subsequent test, the probe trial. Our results suggest that the answer to this question is yes. Zebrafish exposed to a particular tunnel open during the free exploration (training) phase of the maze showed a side preference in the probe trial that corresponded to this prior exploratory

Acknowledgments

Supported by NIH/NIAAA (USA) and NSERC (Canada) grants to RG and by the University of Oviedo (Spain) visiting grant to LMG-L. We would like to thank Ryan Hoffman for conducting pilot studies and constructing the maze.

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