The neurotransmitter systems relevant for motor control and movement disorders are very similar in zebrafish and mammals. Thus, the major biogenic amine systems are well conserved and the CNS domains, which harbor cell body groups, are similar, with a few exceptions (27). Contrary to invertebrates such as D. melanogaster and C. elegans, where receptor mechanisms are very different from mammals despite the similar nature of many neurotransmitters, the zebrafish neurotransmitter receptors are in general very alike those of mammals. Genome duplication has resulted in expansions of some receptor families, and the total number of G-protein-coupled receptors in zebrafish is larger than that of mammals (for a detailed description, see (27)). For example, the three α₂ receptor subtypes (α2a–c) are similar to those of mammals, but there is a fourth duplicated receptor that is not found in mammals (α₂da and αdb) (28, 29). The potentially duplicated α₂a–c receptors have been lost during zebrafish evolution. Genes corresponding to the three histamine receptors (hrh1–3), known to be important in mammalian brain functions (30), are found in zebrafish and expressed in the brain (31), and their ligands have significant effects on zebrafish locomotor activity.

The rate-limiting enzyme tyrosine hydroxylase (TH) is duplicated in zebrafish (32), and this phenomenon has not been noted in most papers that deal with the zebrafish dopaminergic system. Thus, most studies which deal with neurotoxicological or genetic manipulation of the dopamine system, only TH1 has been examined, since also available antibodies detect this form efficiently (33). There is now evidence that TH2 expressing neurons, in addition to those which express TH1, are affected by translation inhibition of PINK1, a mitochondrial kinase important in autosomal hereditary Parkinson’s disease (34). The expression patterns of TH1 and TH2 are complementary. TH2 is expressed in four major bilateral clusters in the preoptic region and hypothalamus (33), whereas a total of 17 cell groups in the whole brain express TH1 (17). Recently, we have developed an antibody against TH2, which shows that the TH2-expressing cells produce TH protein and the cells display a distinct morphology (Semenova et al., in preparation).

Whereas mammals have two distinct monoamine oxidase genes, the zebrafish genome encodes for one enzyme which appears functionally as a hybrid of the two mammalian forms (35), with eight of the 12 residues of the active site identical with human MAO A and MAO B, two with human MAO A and two appear unique for zebrafish (35). MAO mRNA is expressed early (at 24 hpf) in developing zebrafish, and enzyme activity is measurable at 42 hpf (36). Inhibition of zebrafish MAO with deprenyl strongly elevates brain 5-HT content and decreases swimming activity sharply in a dose-dependent manner (36). This effect is due to release and uptake of serotonin in non-serotonergic neurons in basal hypothalamus (36). It is important to take this into account in, e.g., toxicology experiments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which require conversion to 1-methyl-4-phenylpyridinium (MPP+) by MAO to induce a decline in motility in both larval (17) and adult (37) zebrafish.

The cholinergic neurons in zebrafish brain can be found in the telencephalon, preoptic region, dorsal thalamus, pretectal nuclei, hypothalamus, optic tectum, and tegmentum (38–40).

The brainstem of zebrafish larvae contains approximately 150 reticulospinal neurons that project to spinal cord to regulate execution of an array of different behaviors and movement patterns. Of these reticulospinal neurons, the Mauthner neuron, discovered in 1859 by Ludwig Mauthner, is the most famous and is thought to be the sole driver of the escape response, which also is called the startle response. The startle response of freely moving larval zebrafish has been characterized and elegantly demonstrated to include a short latency and a long latency startle response (21). Both types of startle response are short lasting and completed in less than 1 s after stimulation. Laser ablation of the Mauthner neuron abolished the short latency startle response, whereas the long latency startle responses were unaltered, indicating that only the short latency startle response is mediated by the Mauthner neuron. Other neurons of the brainstem such as MID2cm (Mauthner neuron homologue, middle dorsal 2 contralateral medial longitudinal fasciculus interneuron in rhombomere 5) and MID3cm (Mauthner neuron homologue, middle dorsal 3 contralateral medial longitudinal fasciculus interneuron in rhombomere 6) cooperate with the Mauthner neuron to provide the escape response with a direction (22). Stimulation of the head versus the tail elicits activity in a specific but different manner in Mauthner neurons and MID2cm and/or MID3cm neurons. In the case of a head stimulus both Mauthner neuron and MID2cm or MID3 neuron respond, whereas when the tail is stimulated only the Mauthner neurons are activated. Orger et al. (23) studied the role of specific reticulospinal neurons in turning behaviors and slow swimming, and found forward-preferring neurons among the neurons of the medial longitudinal fasciculus, nucMLF. In the same study, neurons that execute right or left movements were also identified. A schematic drawing of the anatomical position of the described neurons is presented in Fig. 2, and also their connections via commisural interneurons to lower motor neurons in the spinal cord are illustrated.

Zebrafish larvae show spontaneous movement as early as 17 hpf (2). At 3 dpf, the fish show bursts of activity, but the continuous beat-and-glide swim pattern is observed at the earliest at 4 dpf. As the fish grows older the time spent swimming increases. By 5 dpf larvae have almost consumed the nutrients in their yolk sack and need to start hunting for food in order to survive. At this stage different behaviors can be observed, such as prey capture (41) and sleep (42, 43).

In order to link neurons to circuits and behaviors, behaviors can be dissected into smaller units or movement patterns. Kinematic analysis, which includes dissection of specific behaviors into different movement patterns, provides a detailed description of the repertoire of movements that the zebrafish combines to produce specific behaviors. For dissecting the kinematics, it is essential to observe different behaviors or movement patterns of zebrafish using high frequency imaging (starting at 500–1,000 frames/s). Behaviors that need to be assessed with high frequency imaging are very rapid, lasting only for milliseconds, e.g., startle response and prey capture. For other movement patterns and behaviors such as place preference, sleep, or motility assays, which can be followed for long periods (from seconds to days), live imaging with standard video tracking frequency (starting at five samples/s or less) is adequate.

Several studies have addressed the movement pattern repertoire of zebrafish larvae and classified the movement patterns in different manner (41, 44, 45). Zebrafish exhibits an escape response consisting of a C-start turn (a Mauthner cell-driven activation of motor neurons on the contralateral side of the spinal cord enabling the fish to turn and swim away from the stimuli) in response to acoustic/vibrational stimuli (21) and O-bends (a Mauthner cell-independent movement that is slower than the C-start turn) in response to dark flashes (46). For navigational purposes, adjustment moves, turns, and scoots (45), and slow-swim-like patterns with the pectoral fins can be detected (41). These movement patterns can be detected with high frequency imaging, which requires specific cameras that can acquire images at a high speed (500–1,000 frames/s) and lenses with resolution to image larvae. Strong light, either the infrared or the visual light wavelength range, is also needed as the contrast between the larvae and background needs to be high enough to distinguish the larvae. As the behaviors that are detected are very rapid, it is an advantage if the stimuli and recording of the behavior can be synchronized. Several individuals can be recorded in the same group, and when the images have been captured, the different movement patterns can be identified from the images.

Above, we already reviewed how the escape response is divided into two different startle responses. Another robust response of the larval zebrafish is the prey capture behavior, which consists of combining the movement patterns described above. Paramecia are often used as live food for rearing zebrafish, and the prey capture behavior of zebrafish larvae has been studied by high frequency imaging when exposing paramecia to the fish (41). As a consequence, the larvae orient themselves in response to the position of the paramecia by small discrete movements, including routine-like turns to align the body axis of the larvae with the paramecia and slow swim-like patterns with the fins to slowly move closer to the prey before engulfing it (41). Prey capture has been shown to be mediated by two specific neurons as bilateral ablation of two neurons, MeLc and MeLr, in the medial longitudinal fasciculus (nucMLF) completely abolished the prey capture without affecting other behaviors (47). Recently, filtering of small visual cues in the optic tectum was demonstrated to be essential to enable prey capture in zebrafish larvae (13).

Motility assays are the most common behavioral assays used for both larval and adult zebrafish. We and other groups have established methods for detecting the movement and motor functions of both adult (37, 48, 49) and larval zebrafish (17, 26, 31, 36). We have used a computerized video tracking system successfully for almost a decade. A number of modifications have been done to the basic setup in order to observe different aspects of zebrafish behavior. Automated video tracking that converts the live image to graphical representations allows quantification of the movement of fish. In this manner, the behavior of larval and adult zebrafish can be monitored from seconds to days. If the tracking lasts for several days, it is of utmost importance that the conditions, noise, light, humidity, and temperature of the observation room is held at constant and appropriate levels. The basic setup is robust and relatively simple, utilizing both wavelengths from the visual range and/or infrared light for detection of the freely moving pigmented zebrafish. Furthermore, this system offers high throughput, because as many as 96 larvae can be observed simultaneously in a single system.

We have focused on the locomotor functions of zebrafish, such as total distance moved, velocity and frequency of movement. We have also observed the circular movement of zebrafish, such as turn angle, angular velocity, and meander (31, 36, 37, 48). Furthermore, we have assessed behaviors such as dark flash response in larval zebrafish (Sundvik et al., in preparation) and place preference in both adult (37, 48) and larval zebrafish (unpublished observation).

The locomotor behavioral method with its several modifications, has limitations that have to be taken into account, but can easily be mastered. In the following, we deal with the major variables. To start with, it is important to take into account how many individuals are necessary to be included in the experiment, and if both the control and treatment group should be monitored simultaneously or if they can be analyzed subsequently. If a large number of individuals per group is essential, this reduces the number of parameters that can be assessed, as the well size is reduced. If place preference is a priority parameter, it is important to have arenas spacious enough for the fish to be able to show a preference. Usually, for larval zebrafish, we used standard well plates of different size, ranging from 6- to 96-well plates, which result in arena diameter of 36–7 mm, respectively. Most often, we have used the 48-well plates, with arena diameter of 13 mm, as the different parameters of movement and different behaviors can still be distinguished. This format also offers high throughput as the behavior of 48 larvae can be assessed simultaneously. The standard well plates have one major drawback, as they are made of transparent instead of white plastic, the inner surface of the wells need to be modified in order to inhibit formation of mirror images of the larvae on the walls of the wells. Therefore, we have also made arenas of white plastic (this offers better contrast between the larvae and background and does not produce mirror images of the larvae) and have a larger diameter (40 mm) where the place preference behavior of zebrafish larvae is easily monitored. When assessing movement and place preference in adult zebrafish, the same basic principles as for larvae apply. It is important to have spacious enough arenas, the animals should be easily distinguishable from the background, and the method should offer options for tracking the behaviors of several individuals simultaneously. As the adult zebrafish tend to jump, it is important that the walls of the arenas are high enough (at least 8 cm over water level) so that the fish cannot move from one arena to the other. The last aspect is that the tracking systems that utilize only one camera create a two-dimensional representation of the movement pattern of the fish. Therefore, the depth of the water column in which the fish is swimming should be as small as possible when horizontal movement is tracked. Recently, some groups have reported that the anxiolytic effect of drugs can be assessed by observing whether the fish spend time in the upper compartment of the water column or at the bottom of the tank (50). Taken together, our approach for assessing behavior offers a robust and crude measurement of motor functions in developing and adult zebrafish.

Apart from studying the locomotor activity, it is feasible both in larval and adult zebrafish to assess the response the fish show to changes in the environment. This can either be studied by moving white and black stripes unidirectionally under the arena where freely moving fish are held, to analyze optomotor response, or by fixing the zebrafish with, e.g., agar and monitor eye movements (optokinetic response). These two assays have successfully been used to screen for visual system defect mutants (51, 52).

Fifteen years ago, the large-scale screen in Tübingen identified 166 zebrafish mutants with abnormal movement (44). The identified mutants that were identified by an abnormal touch response fell into 14 phenotypical groups affecting 48 genes in total, of which 18 genes affected somatic muscle development and the remaining 30 genes were suggested to affect neuronal development. The latter group comprised mutants with reduced motility, circling behavior, and motor circuit deficits. A circling behavior mutant, twitch twice, was identified in this screen as a mutant that only showed right or left turns, rather than alternating turning behavior to both sides of the trunk as wild-type zebrafish to accomplish straight-forward locomotion. The twitch twice mutant carries a mutation in the roundabout receptor family gene, robo3 (53). This class of genes is important for axonal guidance, and in the case of the twitch twice mutant, the axons of the Mauthner neuron did not cross the midline as in normal larvae (53). As a consequence, the Mauthner neuron axons are either on left or right side of the trunk and thereby the larva exhibits a unidirectional behavior. A few years later in another mutagenesis screen, the touchtone mutant was identified. This mutant showed reduced motility and pigmentation defects, and the mutation was shown to be located in the transient receptor potential melastatin 7 (trpm7) gene (54). TRPM7 is an ion channel permeable for Mg²⁺ and Ca²⁺ and widely expressed in murine cells. Interestingly, loss of trpm7 has been correlated to parkinsonian dementia (55). In zebrafish trpm7 mutants the pigmentation deficit was induced by necrosis of melanophores. The cell death could be inhibited by inhibition of melanin synthesis. As melanin and catecholamines share the same metabolic pathway, this finding gives new insight about the mechanisms underlying cell death of pigmented neurons and can offer a new approach for protecting pigmented neurons from cell death.