Research reportAnalyzing habituation responses to novelty in zebrafish (Danio rerio)
Introduction
As the simplest form of learning, habituation has long been used in biological psychiatry research to examine animal cognitive phenotypes and their alterations produced by different experimental manipulations [1], [2]. Habituation to novelty represents attenuation of innate behaviors, as subjects become accustomed to the environment [3], [4]. Intra-session habituation reflects spatial working memory, whereas inter-session habituation is commonly used to assess middle- and long-term spatial memory [6]. In addition, habituation may represent deeper neurobiological constructs, such as adaptive processing of sensory information [7] and development of a cognitive map [5]. Impaired habituation can also be associated with increased anxiety [2], thereby providing valuable insight into anxiety-memory interplay [8], [9], [10].
Habituation has been extensively studied in various rodent experimental models, showing that animal habituation phenotypes are highly sensitive to various pharmacological manipulations [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. For example, rats chronically exposed to fluoxetine, display increased habituation to novel environments [21], [23], whereas another agent with anxiolytic properties, morphine, appears to impair mouse habituation [11], [18], [24], [25]. Although ethanol stimulates locomotion in mice and rats [24], [26], [27], its repeated administration impairs animal habituation [20], [26], as do the psychostimulant/anxiogenic drug caffeine [12], [24] or kindling with low doses of the anxiogenic agent pentylenetetrazole (PTZ) [16], [17], [22].
With immense genetic homology to humans and rodents, zebrafish (Danio rerio) present a highly efficacious model for human disorders [28], [29]. In addition, zebrafish have proven to be beneficial in the study of behavior and its modulation by various endo- and exogenous factors [29], [30], [31], [32], [33], [34]. Despite being widely studied in various rodents [1], [3], [4], [26], [35], [36], habituation has not been extensively evaluated in zebrafish–a model species that is becoming increasingly popular in neurobehavioral research [29], [33], [37], [38], [39], [40], [41].
Until recently, fish behavior was generally assumed to be instinctively driven, with little cognitive ability (rev. in Ref. [42]). However, it is currently known that fish are capable of forming spatial memories and cognitive maps [42], [43], providing an opportunity to explore their habituation behaviors in depth. Several recent studies suggest that zebrafish can habituate to various stimuli, including conditioned place preference [44], [45], light/dark locomotion [46] and the startle reflex [47], [48] testing. Novelty-based paradigms are commonly used in behavioral neuroscience to study both affective (e.g., fear, anxiety) [35], [49] and cognitive (e.g., habituation) [1], [3], [4], [36] phenomena. Since relatively little is known about adult zebrafish habituation, this study aimed to characterize their habituation to novelty in detail, and to assess its sensitivity to different experimental manipulations.
Section snippets
Animals and housing
A total of 210 adult (3–5-month-old; ≈50:50 male:female ratio) wild type short-fin zebrafish were used in this study. The animals were obtained from local commercial distributors (Petco, Rockville, MD and 50 Fathoms, Metairie, LA) and given at least 20 days to acclimate to the animal facility. The fish were housed in groups of approximately 20–30 fish per 40-L tank. All tanks were filled with deionized water, with room and water temperatures maintained at ≈25 °C and water pH at 7.0–8.0.
Habituation trials
In 6-min trials, we found significant increases in exploratory behavior and decreases in freezing behavior over time (Fig. 1A). One-way ANOVA revealed significant time effects for transitions to top (F(1,37) = 45.263, P < 0.0005), time in top (F(1,37) = 44.801, P < 0.0005), erratic movements (F(1,37) = 21.355, P < 0.0005), freezing bouts (F(1,37) = 7.246, P < 0.05), and freezing duration (F(1,37) = 8.798, P < 0.005). In addition, significant differences were found between first and last 3-min intervals for
Discussion
Although habituation is traditionally utilized to examine rodent exploration and cognition [36], [54], [55], it has not been comprehensively evaluated in adult zebrafish. This study was the first in-depth systematic analysis of habituation phenotypes, attempting to provide insights into zebrafish responses to spatial novelty.
To establish zebrafish model of habituation, we first analyzed intra-session habituation using both short (6-min) and long (30-min) trials, and showed steady increases in
Acknowledgements
The authors thank M. Strong, H. Badani and J. DiLeo for their help with this project. The study was supported by NARSAD YI award (AVK), Tulane University Neuroscience Program's and Gordon Fellowships (DT), Provost's Scholarly Enrichment Fund (BB and JT), Lurcy Fellowships (BB, DT, and JT), as well as by Newcomb Fellows Grant and Tulane University Intramural Research Funds (AVK).
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