15.1. INTRODUCTION

Models are used to represent complex problems in simplified forms—physics, chemistry, and biology all make good use of models. The most familiar are the mathematical sorts that form the basis of natural science theory. In the life sciences, the concept of modeling can extend further to include experimental procedures and nonhuman subjects. For example, a neuroscientist might employ a rat running in a radial-arm maze to study working memory processes, or a mouse in an open-field test to study anxiety. The value of a model is primarily a function of its fidelity: in the case of a theoretical model, fidelity is measured in terms of predicted findings; in the case of biological models, the issue is couched in terms of validity. It is this second kind of model that concerns us in this chapter on neuroscience methods, where the challenge of model species is particularly acute because behavioral and brain processes are both extraordinarily complex, and the problem is to find species that display both interesting behavior and easily accessible neural processes.

Rats and mice, unquestionably the most successful models in neuroscience, have been extremely effective in helping determine which mammalian brain regions and neurotransmitter systems involved in cognition, learning, and other varieties of behavioral function. But the invertebrate Aplysia, a marine mollusk, has also served as a molecular model of memory processes [1]. Such seemingly unrelated model species are useful to the extent that they balance external validity, simplicity, and cost. Most recently these considerations have led researchers in behavioral neuroscience to use fish, a sort of middle ground between rodents and mollusks. In this chapter we review progress in the behavioral neuroscience of the diminutive zebrafish (Danio rerio), a species that has already firmly established itself as a model of vertebrate development, and now opens new doors for the investigation of brain mechanisms.

Zebrafish are sometimes identified as an alternative model (relative to classic rodent models), but the term complementary model might be more appropriate since it addresses the use of fish in addition to classic mammalian models. Some questions, such as about the role of frontal cortical and hippocampal structures in learning and memory, cannot be studied with fish since these are not evident (but see [2]). But other attributes of fish make them valuable models in behavioral neuroscience research. Developmental processes can be continuously visualized in species that have a clear chorion (egg sack). Reporter systems can highlight specific neural systems so that their proliferation, differentiation, migration, and projections can be easily discerned. Reversible genetic suppression through the morpholino technique can determine the importance of specific molecular mechanisms for neurodevelopment. Numerous mutants available also help with the evaluation of molecular mechanisms throughout life. Finally, fish are easily bred in great numbers and develop rapidly, reducing the cost of experimentation and significantly increasing research throughput—potentially, more experiments can be run in less time to answer any number of questions.

The merit of fish models is now a matter of record. Zebrafish, in particular, have been well used in genetics, neuroscience, pharmacology, and toxicology (e.g., see [3–7]). The next and ongoing step is to extend the zebrafish model to pursue questions of behavioral neuroscience, an undertaking that requires valid, reliable, and efficient methods of behavioral assessment.

15.2. USE OF FISH MODELS IN BEHAVIORAL NEUROSCIENCE

Fish are the obvious ancestral form of existing tetrapods, so it is not terribly surprising to find that they show most of the behavior seen in terrestrial species in some form or other. In social behavior alone, there are species known to show monogamous mating for life (e.g., angelfish [8]), individual recognition of conspecifics by sight or odor [9], socially mediated learning [10], intricate mate-selection strategies [11], ritualized displays of aggression [12], and communication of danger [13,14]. With respect to cognition and adaptive behavior, fish show highly developed spatial navigation abilities [15], nonassociative learning such as habituation [16,17], precise timing abilities [18–20], Pavlovian conditioning (e.g., see [21]), operant behavior motivated by aversive stimuli such as shuttle box behavior [22], negatively reinforced avoidance [23], and food-reinforced lever pressing positively reinforced responding [24]. In terms of sensory processes, fish have excellent color vision; [25] some species generate and detect weak electrical currents, a sense that they use to detect predators and prey; [26] and have lateral-line organs that allow them to resolve the location, size, and features of distal objects by sensing their pressure shadows.

Behavioral research with fish began with ethologists and comparative psychologists asking questions about the evolution of learning, cognition [27–29], and brain function [30–33]. As in other species, the understanding of the teleost brain has been driven in large part by the development of appropriate behavioral assays (e.g., see [31]). The extent to which basic behavioral and brain processes in mammals and fish are analogous remains an open question—there are clear similarities and differences—and, as with all animal models, the validity of a fish model hinges on the particular question being asked. Many species of fish have been used in models of cognitive impairment, for example, the Japanese medaka (Oryzias latipes) is being used in toxicological studies on effects of the insecticide diazinon (e.g., see [34]), and walleye (Stizostedion vitreum) have been used to demonstrate the adverse impacts of insecticides on cho-linergic systems [35]. Goldfish (Carassius auratus) have historically been used to study learning and memory processes [26,36,37]. Fish have not been widely used in pharmacology but there is no reason to believe that they would not be suitable (e.g., see [3]).

Zebrafish have rapidly become a prominent model for studying the molecular basis of vertebrate neurodevelopment [4,38,39]. The scientific potential of the zebrafish was discovered by George Streisinger [40]. The clear chorion of the zebrafish allows continuous visualization of neuroanatomy; their rapid development and accessibility to genetic analysis make the zebrafish an excellent model system for molecular and mechanistic studies of neurodevelopment. Since its introduction, many genetic mutants have become available, including varieties that can help determine the molecular mechanisms of neurobehavioral function. More recently, the availability of morpholino techniques, whereby specific parts of the genome can be reversibly suppressed during early development, provides a unique way to explore the molecular biology of development. Zebrafish have been critical in the identification of a variety of genes affecting various aspects of neural development and function (e.g., see partial list in [41]). As a result, the genetics and physiology of learning and memory are now being more widely studied in zebrafish (e.g., see [42]). Many tasks are now able to tap behavioral processes previously only studied with rodents and goldfish [3,25,43–48].

15.3. PROCEDURES AND PROCESSES

It might seem a simple matter to develop a valid battery of behavioral tests to study learning and cognition in fish, but it has not been so. One problem is translating between terrestrial and aquatic habitats; another problem is finding reliable and valid dependent measures; nor are theorists always in agreement about how to classify behavioral processes [49]. A somewhat simpler question involves distinguishing between procedures: those that involve stimulus presentations (e.g., to study reflexes and fixed-action patterns), and those that involve arranging consequences for behavior (e.g., to study instrumental or operant behavior). All other behavioral preparations derive from these—Pavlovian conditioning involves signaling a stimulus presentation (e.g., a tone that signals a shock), and operant discrimination involves signaling a consequence (e.g., a color that signals which arm of a maze contains food).

Further subtle variants of these basic procedures can answer any number of questions about processes; [50] for example, a rat’s visual contrast sensitivity can be tested with great accuracy by arranging an operant discrimination between sin-wave gratings and gray patches [51]. Four cases of apparatuses adapted to study behavioral processes in zebrafish are illustrated in Figure 15.1. At the top left (1) is an aquatic version of the T-maze, an operant task used to study problems in discrimination including attention, memory, and reinforcement. In the case of fish, the T-maze has been used to study color discrimination in zebrafish [25], problems in navigation (e.g., in goldfish [52]), and effects of genetic manipulations on habitat selection as in the maze shown here in which the dependent measure was latency to reach the favorable habitat [3]. The top-right illustration (2) depicts a rotating drum apparatus that has been used to study reflexive escape (a variant of the opto-kinetic reflex test). In this test, a typical fish will flee the rotating band (a) by hiding behind the central pole (b), a visually guided escape taxis [3,53]. The lower-left illustration (3) depicts a setup used to study novelty-elicited exploratory behavior. A fish placed in the tank will visit the raised platform through the door (c) to explore a small, submerged stimulus (d) such as a colored bead; [17] exploration shows habituation to familiar stimuli and dishabituation with the introduction of new stimuli. The lower-right illustration (4) depicts an aquatic version of the place-preference procedure used to measure conditioned appetitive stimuli. In this test, a subject is first exposed to an unconditioned stimulus, e.g., cocaine [3], in one of two distinctive halves (e and f) of the tank, and then later with the partition opened, it is given a preference test. In the sections that follow we present these tests and others in greater detail, emphasizing behavioral processes as much as procedures.

FIGURE 15.1

Four apparatuses that have been used to study learning and memory in the zebrafish. (1) T-maze. The T-maze can be used to study a variety of questions in learning and cognition including discrimination [25], and spatial and nonspatial navigation (e.g., (more...)

15.3.1. Assessment of Swimming Activity in Newly Hatched Zebrafish

It is important to determine the motor behavior function in young zebrafish for studies of development as well as for higher throughput tests of the adverse effects of early toxicant exposure. Figure 15.2 (top inset) shows how a dissecting microscope can be used to image the movement of newly hatched zebrafish. Either through manual scoring of videotapes using a grid system or a computerized digital video tracking system, the swimming activity of newly hatched zebrafish can be reliably indexed. The lower graphs in Figure 15.2 shows the significant reduction in swimming activity with 100 ng/mL of chlorpyrifos from fertilization through hatching on day 5 when the behavioral test was conducted on day 6 or day 9 [54].

FIGURE 15.2

Swim test to evaluate motor function in newly hatched zebrafish. The left side of the upper inset (A) shows a video microscope setup with five arenas; the right side (B) shows a close-up of the cylindrical arena and grid pattern used to measure distance (more...)

15.3.2. Reflexes and Habituation

Much research on the physiological mechanisms of zebrafish behavior has focused on sensory-motor development (e.g., vision, swimming, and touch-elicited reflexes) in larvae or young fish (review in [55,56]). The simplest investigations are those of the tap-elicited startle reflex, the so called “C-start” response, which has been found to show an increased latency with early alcohol exposure [57,58]. The development of touch-elicited escape behavior has been detailed by Granato et al. [55]: “Although the embryo is resting most of the time, touching the tail tip induces a fast and straight movement away from the stimulus source. In contrast, mechanical stimuli near the head of the embryo induce a fast escape response, where the embryo turns 180° along its horizontal body axis. At 96 hours the larva is freely swimming, changes swimming directions spontaneously, and is able to direct its swimming towards targets” (p. 399).

We have recently examined habituation of tap-elicited swimming in unrestrained zebrafish to evaluate the effects of toxicants and drugs on a nonassociative learning process. Figure 15.3 (upper inset) illustrates a fully automated procedure using commercially available video tracking software (Ethovision, Noldus, Inc., Wageningen, The Netherlands). Fish were studied individually in a test battery consisting of eight 50-mm diameter tanks. The test arranged a “step up” transition in stimulus rates, with 20 taps presented with an inter-stimulus interval of 10 sec, followed immediately by 20 additional taps with an inter-stimulus interval of 20 sec. The graph depicts the effect of scopolamine (fish were immersed for 5 min in 200 mg/L of scopolamine prior to testing) on swim distance in the 5 sec after each tap (previously unpublished data). Both control and scopolamine-treated fish showed habituation of tap-elicited swimming, but only the control fish showed a reliable recovery in the taps immediately following the “step up” transition, a finding in the scopolamine treated fish that is consistent with a selective disruption in short-term memory (for a theoretical treatment of short- and long-term memory in habituation, see [59]).

FIGURE 15.3

Tap-elicited swim test used to study habituation in the zebrafish. The left side of the upper inset (A) shows a horizontal array of eight arenas below a digital camera; the right side shows the push-solenoid used to deliver sharp taps under the cylindrical (more...)

Zebrafish show a highly developed visually guided escape reflex, which may be related to the opto-kinetic response in other species [53], escaping a stimulus behind a place of concealment. This concealment reflex may be analogous to the targeted response concealment behavior described in mice by Blanchard [60], where if mice are familiarized with a container containing a place of concealment, they flee directly to that place when threatened. Figure 15.1(2) shows an apparatus developed to study this visually guided escape reflex in zebrafish [3,53]. Fish are tested by rotating the outer cylinder of the apparatus, which contains a vertical black band (a), and observing the subject’s orientation with respect to the band and a central cylinder (b) behind which it can hide. The test can be adapted to test visual function [61,62] and has been used to measure visual contrast sensitivity of zebrafish [63].

Exploratory behavior in novel environments has been used to assay anxiety in rodent models (e.g., see [64–66]) and analogous procedures have entered the zebrafish literature. Several experiments show that the fish first explores a stimulus predominantly using the right eye and subsequently approaches the stimulus favoring the left eye [67]. Figure 15.1(3) shows an apparatus employed by Miklosi and Andrew [17] to study lateralization of visual exploration in the zebrafish. Subjects are first trained to visit a box suspended in their home tanks by entering a door (c) to eat. After they reliably enter the box, a colored bead (d) is lowered into the water with the behavior of the fish recorded on video. Right eye use and biting were highly probable the first time a stimulus was presented and declined in probability in two subsequent trials, demonstrating habituation (see also [16,68–70]).

15.3.4. Operant Conditioning and Mazes

Although it might be assumed that this predominance of aversive procedures is because aversive procedures are more rapid than appetitive procedures, there are exceptions such as that shown by Williams et al. [46] who trained fish to alternate between two feeding sites in an average of 14 trials. The task is essentially an appetitive version of the shuttle box discussed above, except that trials are initiated by the experimenter tapping on the center of the tank and 5 sec later dropping a small amount of food in one end of the tank (the location of food is alternated between trials); the dependent measure is the position of the fish immediately before the delivery of food. Carvan et al. [57] used this task to show dose-dependent detrimental effects of ethanol on learning and memory in zebrafish.

Perhaps the earliest example of behavioral research with zebrafish is a maze learning study in which negotiation of a left-right-left-right maze and approach to black or white stimuli was trained by eliciting an anode galvanotaxic reflex that elicited approach to the target stimulus, a procedure that is somewhat difficult to implement [75]. Colwill et al. [25] recently trained color discrimination in zebrafish by placing different colors at the end of each arm of a T-maze (green vs. purple and red vs. blue) and feeding the fish only at one arm. These researchers unambiguously demonstrated discrimination of color by arranging discrimination reversals (i.e., a crossover design) and experimenter-blind testing. In a similar T-maze apparatus as shown in Figure 15.1(1), Darland and Dowling [3] reinforced choice of one arm by providing it with a goal box containing deep water, artificial grass, and marbles (however, the dependent measure was the reduction in latency to reach the enriched arm, a result that could be caused by habituation of fear in the novel maze apparatus).

The three-chamber maze shown in Figure 15.4 was developed by Arthur and Levin [43] to assess learning and memory in the zebrafish. The three-chamber maze can be thought of as a simplification of the T-maze, but one in which aversive consequences follow errors. The start area is the middle “start chamber,” and there are vertically sliding doors on either side of this central start area leading to left and right choice areas. At the outset of a trial the fish is placed in the start chamber and allowed to move about for a brief period. In the choice phase, the vertical sliding doors to the left- and right-choice chambers are opened and the fish is allowed time to swim to one or the other; if it persists in the start chamber, a fish net is waved in the chamber (a “threatening stimulus”) until it makes a choice. After making a choice, both vertical sliding doors are closed. If the choice is correct (i.e., to the goal side) the fish is permitted to swim for a short period of time; if the choice is incorrect the sliding partition is moved to the “restricting position” for a short period of time. This procedure is repeated for a fixed number of trials. Dependent measures in the three-chamber shuttle maze include latency to escape the start chamber and correct choices [7,43]. Initial tests with the maze [43] showed that zebrafish could be trained to turn in a particular direction (spatial learning) or to approach a particular color regardless of location (nonspatial learning).

FIGURE 15.4

Learning and memory assessment in zebrafish with the three-chamber shuttle maze [7]. As shown in the top inset (A), trials begin with the fish in the “start chamber”; during a choice phase, both vertical sliding doors are opened. After (more...)

We have used the three-chamber maze to show that the delayed spatial alternation behavior is a sensitive index of the persisting cognitive impairment caused by developmental exposure to the organophosphate pesticide chlorpyrifos (Figure 15.5) [7]. A parallel line of investigation (Figure 15.6) [76] found that acute nicotine administration causes a significant improvement in delayed spatial alternation at low doses but impairs performance at high doses. The biphasic effect of nicotine improving memory function at low doses and having less improvement at higher doses is a common finding across a wide variety of species including rats, mice, monkeys, and humans [77–79]. The fact that the same effect was seen in zebrafish points to similarities of nicotinic effects on memory with mammalian species. This similarity can be advantageous because molecular studies of neural function can be more easily studied in zebrafish than mammals. The inexpensive zebrafish and a rapid, short, six-trial spatial discrimination test was useful in determining the time-effect function for nicotine-induced accuracy improvements.

FIGURE 15.5

Early exposure to chlorpyrifos produces a persisting effect on delayed spatial alternation in zebrafish tested in the three-chamber shuttle maze [7]. Both 10 ng/mL (p < 0.05) and 100 ng/mL (p < 0.01) of chlorpyrifos during the first 5 (more...)

FIGURE 15.6

Effects of acute nicotine on zebrafish performance in the three-chamber maze. The top graph presents findings showing that nicotine produces a significant (p < 0.005) linear dose-effect function on spatial alternation: low doses facilitate performance (more...)

15.3.5. Testing Anxiety and Stress Response

Anxiety and stress response can be tested in zebrafish by indexing their diving in a novel tank—many fish, including the zebrafish, show a defensive diving response in response to threat. When zebrafish are placed in a novel tank they tend to dive to the bottom of the tank, dwelling there and gradually rising to the upper levels over a period of minutes. This is similar to thigmotaxis or time spent near the walls of an open field in rodents. We have developed an automated assessment of the diving response using digital video tracking, illustrated in Figure 15.7 (upper inset). The graph in Figure 15.7 shows results of a recent study examining the acute nicotine effect on the stress-diving response [80]. Control zebrafish stayed in the bottom third of the tank for nearly all of the first minute in a novel tank, and then over the rest of the 5-min session spent progressively less time in the bottom. Acute nicotine treatment significantly reduced this effect with 50 mg/mL significantly reducing the initial diving and 100 mg/mL eliminating preference for the bottom. Interestingly, this effect of nicotine on the diving response did not seem to be caused by drug-induced confusion since the same dose significantly improved accuracy in the maze test. In addition, nicotine effects on swimming activity were also congruent with the changes in bottom dwelling.

FIGURE 15.7

A zebrafish diving response test used to measure fear—stress response—to a novel environment. The top inset shows an array of tanks used to study the novelty-induced diving response in the zebrafish. In the study, fish are individually (more...)

15.4. CONCLUSIONS

Despite the small size of the zebrafish, a number of promising behavioral assays have appeared in the literature—it is now clear that the zebrafish model of development can be used in studies of learning, memory, and cognition. There are both appetitive and aversive techniques, and they test a range of behavior, from simple reflexes [81] and fear conditioning [72], to visual discrimination [25] and spatial orientation [43].

In the development and use of animal models of behavioral dysfunction, it is important to develop complementary models to take advantage of the unique advantages of the different species. Non-mammalian vertebrates such as zebrafish provide the opportunity to directly observe neurodevelopmental processes and determine the impact of developmental permutations on learning and memory. Zebrafish are particularly valuable because of the availability of morpholino techniques to transiently suppress specific parts of genomic expression. The development of new methods for high-throughput tests of cognitive function for fish can provide means for rapid screening of potential toxic agents as well as promising therapeutic agents. It is equally important to develop specific tests of various aspects of cognitive function, including habituation, associative learning, memory, and attention, as well as to be able to differentiate changes in sensorimotor function from cognition. Key in the use of zebrafish models is the determination of which mechanisms of cognitive function are similar to mammals and which are different. Non-mammalian models can be used in concert with classic mammalian models to determine the neural bases of cognitive function and discovery of toxicants and potential therapeutic agents.

ACKNOWLEDGMENT

Research was supported by NIH ES10356.

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