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Pierre L. Roubertoux (ed.)Organism Models of Autism Spectrum DisordersNeuromethods10010.1007/978-1-4939-2250-5_1212. Zebrafish Social Behavior Testing in Developmental Brain Disorders
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Abstract
Zebrafish are still a relatively new, but promising, model organism and increasingly used in behavioral studies. This chapter reviews some of the behavioral paradigms that show promise in behavioral neuroscience as it applies to pervasive developmental disorders (PDD) of the brain, such as autism. Predictive and high-throughput animal models of cognition and behavior are increasingly more important in translational neuroscience research and zebrafish could be an excellent tool in this area. The zebrafish’ complex behavioral repertoire, its size, relative low cost, short generation time, and high homology with higher vertebrates make it an excellent model organism. Rodents have traditionally been used to study complex cognitive phenotypes. However, it is particularly the physical features of zebrafish (small size, short generation time) that make it an appealing model to complement the progress made in the rodent literature. Taking into account that no single animal model can recapitulate all aspects of human behaviors impaired in pervasive brain disorders, using zebrafish to complement existing efforts using different models may open up new avenues of discovery not previously explored. While promising, zebrafish are a new model and many existing paradigms are being translated to the zebrafish larval and adult model. This chapter aims to discuss various important behavioral paradigms and how they relate to the study of PDDs.
Key words
Behavioral paradigmsZebrafishHigh-throughput screeningPervasive brain disordersAutism1 Introduction
Zebrafish (Danio rerio) are a small freshwater tropical teleost fish that was first introduced to the laboratory by the late George Streisinger in the 1970s [1]. Zebrafish are diploid and vertebrates that show a balance of simplicity and complexity as model. C. elegans and Drosophila are known for their simplicity and are popular models in behavioral neuroscience, but do not offer the same benefits found in vertebrates such as mice and rats. Zebrafish are thought to straddle both of these groups by offering a vertebrate system with the simplicities of a small, prolific, organism. As such, zebrafish are an increasing complement to other vertebrate models [2], particularly in translational studies that benefit from using a vertebrate model. Zebrafish’ ease of genetic manipulation and fully characterized genome also contribute to its growing popularity.
From an acquisition and maintenance perspective, zebrafish are readily available, inexpensive, and easy to care for in large numbers. They are also highly prolific under appropriate circumstances [3]. Females spawn year round and are able to reproduce every couple of days, producing clutches that may contain several hundred eggs. Zebrafish eggs are large (~0.7 mm at fertilization), optically transparent, and easily manipulated. Eggs are also permeable to a range of pharmacological agents. Generation time is relatively short, at approximately 3 months. This allows zebrafish to be used for selection experiments. Compared to rats and mice, zebrafish are economically more feasible to be used in high-throughput screening studies [4].
Adult zebrafish provide substantial behavioral and system complexity, as well as homology to higher vertebrates [5]. The combination of genetic techniques available and the physiological and genetic features of zebrafish make them a particularly attractive model organism for many human conditions. Zebrafish organs are functionally and morphologically similar to those in humans, and mutations affecting functional processes may give more insights into pathways and mechanisms relevant to human conditions [6]. Compared to Drosophila and C. elegans zebrafish also show far greater similarities to the human neurological system [2]. Zebrafish are behaviorally complex and display a rich behavioral repertoire (extensively summarized by [7]. At the larval stage zebrafish already display behavioral outputs that can be exploited [8]. This allows for testing early in development, and throughout the life span. Zebrafish are also a highly social species and spend the majority of their time in shoals. Shoaling tendencies commence at a relatively young age, and continue to develop over time [9].
Zebrafish have been suggested as a model to study pervasive developmental brain disorders such as autism spectrum disorder (ASD) [10, 11]. Understanding the pathogenesis of complex behavioral disorders is a major challenge in biomedical research. Zebrafish offer an opportunity to improve our understanding of basic brain function through behavioral screening. Zebrafish can be a tool to study in vivo function of genes possibly implicated in pervasive brain disorders: the larvae develop rapidly and externally and a large number of larvae can be generated and used for molecular and genetic screens. Brain development can be observed at a single-cell resolution while the larvae are still transparent [2, 10, 12]. At this stage neurons can be ablated or manipulated using lasers, fluorescence microscopy, and light-triggered ion channels [13], further enriching the possibilities for investigation.
While many of these developmental brain disorders have also been modelled in rodents, zebrafish offer yet another perspective and in addition to the attractive physical characteristics described above, also the possibility of large-scale screening studies at a fraction of the cost of the equivalent using rodents.
Behavioral studies can contribute to the understanding of many neurological disorders that feature behavioral symptoms. Behavioral models that have previously been developed for mammalian models can, in most cases, be applied to zebrafish with no or minor modifications [9, 12]. Zebrafish behavioral research is still not as established as in other models, and while zebrafish do not display complex grooming behavior, it do display other behavioral stereotypies as repetitive thigmotactic (swimming near the walls of an enclosure) and circling behavior. Alterations in learning and memory performance, motor function, or social behavior can provide insight into a range of neurological conditions [14]. For example, loss of function was confirmed through behavioral testing with zebrafish as a model for Parkinson’s disease [15]. Other behavioral studies have investigated stress and anxiety using zebrafish [16]. Zebrafish provide an opportunity to investigate mechanisms behind neurodegeneration and other neurological conditions, increasing our understanding and possibly helping in the development of interventions.
Models such as the zebrafish will not replace clinical research, but are valuable in many subdisciplines, from investigating the function of target genes in particular brain regions to understanding the effects of teratogenic insults throughout development. Particular strengths of zebrafish as a model organism relate to several key characteristics: the similarity between zebrafish and other vertebrates in broad respect, including the layout of the brain [10], the neurochemical properties of the brain [17], and in regard to characteristics for many levels of biological organization [16]. The latter includes the nucleotide sequence of genes, and even a decade ago these factors added up to zebrafish being believed to make suitable models for studying brain function [18]. Findings from studies using zebrafish are expected to translate well to humans on an initial preclinical level, and can shed more light on complex human conditions such as complex developmental brain conditions [10] and aid in the preclinical screening of potential drug targets [19].
2 Zebrafish Social Behavior
Zebrafish are a social species that exhibit a preference for conspecifics and start shoaling early on in development and prefer to spend most of their time with conspecifics [9, 20]. Behavioral features of zebrafish are becoming increasingly better characterized [7, 21], such as aggression, fear [21], alarm reaction [22], sleep [17], reward [20], and social behavior [23]. Several groups have made strides in utilizing and characterizing shoaling behavior in zebrafish as well [24]. A number of studies involved labor-intensive and/or subjective characterization of shoal cohesion using, for example, the number of fish present within a cell of a quadrant system [24]. More recently, studies using shoaling have been assessed with video tracking and high-throughput data analysis methods to make these efforts more scalable [25]. Other studies assess shoaling preference as a measure of “closeness” of one test fish to a shoal stimulus of live fish [26], or animated fish images [23, 27].
Different aspects of the zebrafish’ social repertoire may be appropriate for behavioral testing, depending on the condition being modelled. The remainder of this chapter focuses on a series of behavior test paradigms and their uses in zebrafish behavioral neuroscience.
3 Behavioral Paradigms
3.1 Open Tank/Novel Tank Diving Test
The open field is one of the most established and most heavily used tests in animal psychology. This is also a test that requires little adaptation for use with zebrafish and consists of releasing an animal into a plain arena to observe that animal’s behavior over a period of time. Classically, this test has been established using rats and mice, but by creating an area containing water it is just as easily used with zebrafish instead.
The open field test provides an overview, or index, of general behavior but in particular exploratory behavior and thigmotaxis [28, 29]: both crucial responses to novelty. Locomotor activity, spatial learning, and anxiety are all seen altered in developmental brain disorders, such as ASD and attention-deficit/hyperactive disorder (ADHD). The open field is an excellent tool to assess each of these behavioral outputs. Particularly thanks to the larger size of the open field locomotor activity can easily be assessed. In healthy rats, initial response to the open field consists of thigmotaxis and exploratory behavior, which decrease over the time of the trial (habituation to the open field) [28, 29].
The novel tank diving test typically uses a small tank, particularly compared to the open field. Having said that, some groups seem to use the terms interchangeably. Some authors refer to the open field test when using a small tank identical to that often used to house zebrafish [30]. In the rodent literature, the open field is exclusively referred to when using a large open arena that is relatively much larger than the environment in which the animal is housed. Even in relatively large open fields animals scale their behavior according to the arena size [31], and because this behavior is not yet well defined in zebrafish, it is important to clearly define the open field in these cases. The open field or novel tank diving test can both help assess habituation of the animal to the novel environment. Habituation is an evolutionarily conserved adaptive behavior relevant to cognition and several groups have made strides in recording this behavior in zebrafish [32, 33].
Similarly measured in a novel tank is circling behavior. This behavior can be induced by certain psychotropic drugs such as MK-801, ketamine, or PCP, which is similar to the drug-induced circular locomotory behavior seen in autism spectrum disorder (ASD) models in rodents [11]. Zebrafish behavioral output can be modulated with a variety of pharmacological agents [7], often mirroring ASD-related symptoms also modelled in a similar way in rodents. This observation suggests that ASD-related social phenotypes, and likely those associated with other developmental brain disorders, are conserved across various species, supporting the translational value of zebrafish models for these disorders [11].
The procedure used in the open field as well as the novel tank diving test is as follows: one fish is released in the novel environment and allowed to explore it for a set period of time. Different studies have used various time frames, ranging from a few minutes to hours. For the open field, the arena is recorded with an overhead camera and behavior is quantified afterwards by looking at time spent in different areas of the tank. The tank may be divided up into quadrants, or another measure may be used. Endpoints typically recorded are time spent in the center, time spent near the walls of the tank (thigmotaxis) and latency to enter the center of the tank, number of freezing bouts, time spent frozen, distance traveled, average velocity, turning angle, and angular velocity [25, 33].
In the novel tank diving test, the tank used is typically smaller (e.g., 1.5 L), filled with water and divided into 2–3 equal virtual horizontal sections. These may either be demarcated with a line on the outside walls, or demarcated with a computer software tool superimposed on the recorded video image during analysis. In the novel tank diving test the following endpoints are recorded during a 5–10-min session: latency to reach the upper half (or upper third) of the tank, time spent in the bottom of the tank, the center and the top of the tank, the number of entries into the upper portion, the number of erratic movements, freezing bouts and time spent frozen, distance travelled, average velocity, turning angle, and angular velocity [33–35].
The open field test is versatile and heavily used in rodent behavioral research related to neurodevelopmental brain disorders. One study established involvement of the amygdala and hippocampus by using the open field to assess adaptation and habituation after lesioning these areas of the brain in rats [36]. McFarlane et al. demonstrated that the inbred mouse strain BTBR T1tf/J (BTBR) demonstrates multiple behavioral phenotypes relevant to autism, such as reduced social approach, low reciprocal social interactions, and impaired juvenile play when compared to C57BL/6J (B6) controls [37]. The open field was used to measure motor function and exploratory activity, and control for these as potential confounding factors in the social measures. While the above are just some examples of substantial and growing body of rodent literature, strides are being made with the use of zebrafish as well to model neurodevelopmental brain disorders. Zebrafish have already been used to model autism-like behaviors. The drug MK-801 causes impairment of normal zebrafish behavior in the open field, and affects learning and memory in zebrafish [38, 39]. More specifically, it appears to model autism-like behaviors by decreasing social interaction or shoaling in zebrafish [39, 40].
3.1.1 Procedure and Materials
The open field can be used in both juvenile and adult fish, and the novel tank diving test is typically used in studies with adult zebrafish. The exact protocol will be dependent on the requirements of the study, but in general terms the typical setup is as follows:
Zebrafish must be experimentally naïve, and acclimatized to their home environment for at least 10 days prior to testing. Zebrafish are best housed in groups, unless the protocol requires isolation. Groups are randomly assigned to treatment groups, when appropriate. Treatment can consist of exposure to a drug target or other chemical compound, but groups may also be separated by their genetic background when screening gene targets. The groups of fish to be exposed to the shoaling paradigm are transported from their home tank either directly to the experimental environment or to a preexposure tank, if a drug exposure paradigm applies. In which case the suggested preexposure time is 1 h in a separate tank that is sufficiently aerated. Any drug or compound should be fully dissolved in sufficient dechlorinated tank water to hold the complete group of fish for the duration of the preexposure time. After completion of the preexposure, the subjects can be transported in a net from the preexposure tank to the testing arena, if the distance is sufficiently short. To avoid net stress as much as possible it is advisable to keep the exposure or home tank next to the arena without being visible to the fish from within the arena. Depending on the protocol, the testing environment can either consist of only dechlorinated water or it can contain water with the identical drug concentration used in the preexposure period.
Water temperature should remain around 27 °C. Illumination should consist of ceiling-mounted fluorescent lights, which are kept on a regular dark–light cycle (e.g., 12 h on and 12 h off). The tank system must contain aeration and filtration systems, and the water must be dechlorinated for housing and testing tanks. Behavior is best recorded with a front-facing camera that is capable of capturing the entire testing tank in detail. The size of the tank varies among different paradigms in previous studies. As a general rule it is advised to use a tank sufficiently large to provide the subject with enough space to move around while still enabling the capture of detailed behavioral outputs.
3.2 Shoaling
Shoaling is a highly complex social behavior. The DSM-IV lists deficits in social-emotional reciprocity, including failure to initiate or respond to social interactions, as the first symptom of autism spectrum disorder [41]. Modelling social deficits in a highly social species like zebrafish offers a plethora of tools to work with. As described previously, MK-801 administered to zebrafish reduces shoal cohesion [39, 40]. As social deficits are the core symptom in disorders like autism and schizophrenia, this is being investigated as a way to model these human conditions [40].
The size of the open field used to assess shoaling has not proven to alter shoaling behavior in adult zebrafish in one study [9]. And while the size of the open field can vary, various groups use the distance between fish measured in body lengths as a tool to measure shoaling [9, 40]. This allows for a more objective way of measuring shoaling across different studies, perhaps using different arena sizes. Evidence suggests that shoaling commences early on in development, and continues to develop until adulthood [9, 26]. Preference for conspecifics is thought to exist as early as 6 days postfertilization [42], and visual cues are sufficient to evoke this preference [26].
Shoaling has been studied in groups of varying sizes, as well as arenas of varying dimensions. In addition, the measures used to define shoaling have varied greatly across studies. Group sizes range from relatively small with 3–4 zebrafish [40, 43, 44] up to 10 fish [25, 45]. Arena sizes are diverse in size and even shape [9, 35, 40, 44, 46]. More work needs to be conducted to establish if and how arena size and shape may affect shoaling, and more standardized protocols will be helpful for future research.
3.2.1 Procedure and Materials
Shoaling can be studied in both adult and juvenile fish. The exact protocol will depend on the requirements of the study, but in general terms the typical setup follows many of the same guidelines as described for the novel tank diving test: the materials and protocol are the same for housing, water temperature and quality, transportation of fish, and possible preexposure to drug targets.
The exception is that instead of a relatively small tank, a larger arena is used to contain the shoal of fish. Different research groups have used arenas of different sizes in the past, and there is no fixed consensus on optimal arena size. However, it is advisable to provide sufficient space for subjects to distance themselves from the shoal if they choose, and avoid creating an artificial effect of shoaling due to confinement. Having said that, one might also argue that very large arenas promote shoaling as a fear strategy.
Shoaling behavior is best recorded with an overhead mounted camera that is capable of recording the entire testing arena.
4 Social Interaction Testing
Measuring different aspects of social behavior can be a very useful tool in the investigation of developmental brain disorders, such as autism and schizophrenia. Shoaling, as described above, is one such behavioral output used in behavioral neuroscience. Other aspects of social behavior can also be exploited through various social interaction tests. Previously, behavioral paradigms including social interaction testing have been used to investigate targets of interest for autism and schizophrenia in mice [47]. Gene targets involved in these developmental brain disorders have been studied by using knockout mice and social behavior testing [37].