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Zebrafish
Zebrafish J. R. Fetcho, Cornell University, Ithaca, NY, USA ã 2010 Elsevier Ltd. All rights reserved.
Introduction The zebrafish has only recently emerged as a key model for studies of the neuronal and genetic basis of behavior. George Streisinger’s pioneering studies at the University of Oregon established the zebrafish as a genetic model. Although he envisioned the animal as a model for using genetic tools to dissect behavior, for a long time the focus was on using zebrafish for studies of neuronal development. The initial advantages of this animal were its availability (it is a common pet store fish), its transparency, and the abundance of its eggs and larvae (which allowed easy imaging of neuronal development in the live animal), a somewhat short – 3 month – generation time, and some important mutant lines obtained by exposing the fish to mutagenic chemicals. These advantages led to some remarkable developmental/genetic studies by the Oregon group that put the zebrafish on the map as a model organism. Although there were scattered early behavioral studies of zebrafish, two developments growing out of the work in Oregon catalyzed the expansion of zebrafish as a model for behavioral studies. The first was a large-scale screen for mutants done in Tu¨bingen by the Nusslein-Vollhard group. While the focus of this screen was largely developmental and structural, one portion, led by Michael Granato, focused on animals with movement deficits. The isolation of these mutants catalyzed studies directed toward finding the disrupted genes and revealing how they affected patterns of movement. In parallel, the advantages of the transparency of the larval fish for functional studies became clear with work showing that one could load fluorescent calcium indicators into neurons to literally watch active nerve cells light up during behavior in the intact animal. This was the first imaging of neuronal activity with single-cell resolution in an intact vertebrate and raised the possibility of exploring activity patterns in vivo in both normal and mutant lines of fish. The combination of powerful tools at both the genetic and neuronal circuit level made zebrafish unique among the vertebrate models and set the stage for further behavioral studies. While much of the focus on zebrafish behavior has been in laboratory studies, recent work has begun to reveal more about their natural history and ecology. They are members of the Cyprinidae and are found in the flood plains of the Indian subcontinent, where they typically occur in shallow, slow-moving water with visibility 30 cm deep in areas with vegetation, little shade, and a silty bottom. The temperatures over the natural
range of the fish can vary from 6 to 38 C, although they are typically maintained at 28.5 C in the laboratory. The adults are omnivorous, with their food usually consisting of insects and zooplankton. Likely predators include other fish and birds. The fish typically spawn at first light in the wild, as in the lab, with groups of males chasing a female and the eggs becoming scattered upon release. There is some evidence that males protect sites containing gravel substrates that are preferred by females. Such sites may protect eggs from predation and provide them with higher oxygenation. While the studies upon which these observations of the natural biology of the animal are based are increasing, there is still much about features of their biology, such as intra and interspecific interactions, feeding, and habitat choice, that remain to be explored.
Behaviors of Zebrafish Zebrafish show the broad range of behaviors typical of other vertebrates. These extend from more simple sensorymotor reflex responses such as escape behaviors to complex social behaviors such as shoaling, aggression, and mate choice. A major focus of study has been to identify genes that build the primary sensory systems used to assess and respond to the external world including the visual system, the auditory system, the lateral line, and the somatosensory systems. On these fronts, the model has revealed unknown components of the hair cell apparatus for detecting movement in the lateral line and in the ear, as well as genes that are involved in setting up the neuronal and biochemical networks for appropriate visual responses. On the motor output side, contributions of genes to the production of swimming and escape behaviors have been revealed, along with the neuronal organization of these responses. Most of this behavioral work on the zebrafish model has involved laboratory studies of the genetic and neuronal substrates of relatively simple behaviors or parts of behaviors. There is less work dealing with natural variation and behavior in the wild, although this is beginning as well. Similarly, more complex mating and social behaviors have received some attention on the behavioral side, but are only beginning to move to the genetic and neuronal levels. Nonetheless, understanding the genetic and neuronal substrates of natural variation and complex behaviors is impossible without an understanding of the way in which core elements of particular behaviors are built at
633
634
Zebrafish
the genetic and neuronal levels, and there are now very powerful tools that allow attacks on behavior in zebrafish from the genetic through to the neuron and network levels. These are best considered in the light of what is required to achieve an adequate understanding of the proximate mechanisms by which genes and neurons influence the generation of a particular behavior because this is the arena where zebrafish may have the most to contribute as a model.
The Path from Genes to Behavior Genetic Perturbations The influence of genes on behavior is typically revealed by exploring the behavioral effects of perturbation of the activity of those genes. In zebrafish, there are several avenues for such perturbations. The most common is mutagenesis, done via chemical application to adult fish or via viral insertion into the genome to disrupt random genes and screen for behavioral changes. This is especially useful for revealing unknown genes that affect behaviors, but is not trivial as it typically involves large-scale raising of animals to isolate many mutants, sometimes involving many thousands of tanks of fish. A more directed approach uses morpholinos, modified oligonuceotides designed to bind to specific endogenous RNA sequences and disrupt their expression. These are targeted based upon sequence information, so the approach can test possible contributions to behavior of known genes, but cannot reveal novel ones. In addition to these tools, it is now easy to introduce exogenous genes into zebrafish to produce transgenic animals. This can be useful to express particular genes at higher levels by adding an additional copy under a known, possibly stronger than normal, promoter. This approach reveals the consequences of overexpression of a gene. What is currently missing in zebrafish, however, is the ability to remove or replace the endogenous genes by knockouts – a very powerful approach in other standard models for behavioral studies such as mice. Bridging the Gap Between Genes and Behavior The challenge in exploring the influence of genes on behavior is not so much in finding genes that affect behavior, as there are plenty of those in many species, but rather in bridging the gap between the genes and the behavior to reveal how the genes affect neurons and networks and in turn alter behavior. Some of the features of the zebrafish model make it particularly powerful and perhaps the best vertebrate model for bridging the gene to behavior gap. Moving from genes to behavior involves some critical considerations that include finding in which tissues or organs the gene is expressed and what alterations in
neuronal wiring or activity patterns occur following disruption of the gene as, in the end, these changes in the nervous system will alter behavior. Disrupting gene expression or creating overexpression may also cause changes in effector organs such as muscles or in neural connections with muscles, but some of the hardest challenges have been revealing the impact of genetic disruptions on the neuron or network level. The ability to make transgenic zebrafish and the transparency of the larval animal simplifies forging links between genes and neuronal structure and function. By inserting a fluorescent protein under control of the promoter for the gene of interest, the neurons expressing both the fluorescent protein and the protein of interest can be viewed in the intact, living larval fish. This allows direct visualization of which neurons express the gene. Observations of neurons treated in this way in mutant animals or those treated with morpholinos allow detection of changes in neuronal structure related to disruption of the gene of interest. Because disruptions might also occur in cells not expressing the gene, it is important to explore other aspects of neuronal organization as well. There are an increasing number of transgenic lines of zebrafish with different subsets of neurons labeled with different colored fluorescent proteins. These include lines with inhibitory and excitatory nerve cells labeled in different colors to, in effect, provide a color-coded nervous system. By looking at the effects of mutations or morpholinos in these lines, one can better reveal disruptions of neurons and neuronal patterning that might underlie behavioral changes. An additional advantage of zebra fish is the ability to easily examine the activity of labeled neurons, because genetic disruptions of behavior are usually linked to changes in neuronal activity patterns that can result from a mis-wired circuit, changes in the electrical properties of the neurons, or changes in the synaptic connections between the nerve cells. Every modern physiological tool for studying neuronal activity is accessible in the zebrafish model. Conventional approaches to directly recording the electrical activity of individual neurons with glass microelectrodes (such as patch clamp recording) are possible. More unique to zebrafish, the transparency of the larvae allows the imaging of neuronal activity anywhere in the animal. This has been accomplished with fluorescent indicators that sense calcium levels and change their fluorescence upon binding calcium. By loading neurons with such indicators in live fish, the active neurons literally light up during behavior. The availability of genetically encoded calcium indicators has allowed the generation of transgenic lines with built-in optical indicators of neuronal activity, first done among vertebrates in zebrafish. The methods for monitoring activity can be used in transgenic lines with particular cell types labeled, so the function of different subsets of neurons can be examined. These tools
Zebrafish
allow exploration of neuronal activity during behavior at the single cell and neuronal population levels. They are critical to defining both the normal networks underlying behavior as well as the consequences of genetic perturbations that alter behavior. Linking Neurons to Behavior One of the major challenges in studies of the neuronal basis of behavior is causally linking neurons or neuronal classes to particular behaviors. The approach to revealing these links is much the same as that for linking genes to behavior. The activity of the neurons must be perturbed in some way and the consequences for behavior studied. Zebrafish offer spectacular opportunities in this regard because of recently developed optical tools that play to the combination of genetics and transparency that are the strengths of the zebrafish model. Early perturbation studies used lasers focused on nerve cells in the intact animal to kill particular neurons and explore the behavioral consequences. Such removal is useful, but irreversible. Recently, genetically encoded proteins controlled by light were isolated that allow the electrical activity in nerve cells to be turned off or on by shining light on them. These tools take advantage of light activated ion pumps or channels that can affect the electrical activity of neurons. The methods work in zebrafish, and the ability to introduce genes into the fish and focus light anywhere in the nervous system will allow reversible inactivation or activation of different neuron classes to test their contribution to behavior. One can even imagine flashing neurons to turn them on in different patterns to essentially play activity patterns into the nervous system to examine the behavioral consequences of different patterns. The power of these approaches, which are best applied in a transparent animal, is likely to make the zebrafish model even more important in studies of the neuronal basis of behavior.
Examples of the Application of Optical and Genetic Approaches Neuronal Activity During Behavior The utility of zebrafish for behavioral work is best illustrated by examples from many contributions that take advantage of the special strengths of model. Some of the initial work taking advantage of the transparency for functional studies set out to reveal the patterns of neuronal activity in the hindbrain that underlie the escape response – a fast turning movement that fish use to avoid predators. There was good evidence that a set of nerve cells with axons projecting into the spinal cord, including the well-studied Mauthner neuron and other similar, serially repeated neurons in different parts of the
635
hindbrain, were key to generating the escape response, but their activity patterns during behavior were largely unknown. By labeling the neurons with fluorescent calcium indicators, it was possible to reveal differences in which neurons were active, depending on the location of a touch stimulus used to elicit an escape, because different neurons lit up during different escape stimuli. This approach linked patterns of activity in the hindbrain to different forms of a behavior and provided insight into the functional organization of a part of the brain critical to movement. Test of Neuronal Contributions to Behavior by Optical Perturbation The initial tests of neuronal contributions to behavior in zebrafish came from studies in which the contribution of hindbrain neurons active in different forms of escape was tested by laser ablation. This work confirmed in studies ablating individual cells that the different cells in the set of hindbrain neurons including the Mauthner neuron and others like it indeed made different contributions to the escape behavior. Killing particular cells could affect some forms of the escape response and not others, confirming the view that even a seemingly simple behavior can have subtle variations that can only be revealed with studies of an accessible model system. More recent work combined genetic approaches with laser ablations to test the contribution of neurons in spinal cord to movement. This work took advantage of a socalled enhancer trap line in which an insertion of a fluorescent protein into the genome led to the labeling of a very discrete set of spinal interneurons. These inhibitory cells had axons that crossed from one side of the body to the other and were thought to allow a forceful escape bend by blocking activation of muscle on the opposite side of the bend. Removing the neurons, which were easily targeted with a laser in the live animals because they were fluorescently labeled, led to a bilateral muscle contraction and a seriously disrupted escape bend. This directly confirmed the critical role of a class of neuron in a motor behavior. While these two examples focus on laser removal of neurons, the expectation is that future work will use reversible approaches to inactivating neurons with light to allow even more compelling studies of links of neurons to behavior. Genes Affecting Behavior A genetic change can produce a behavioral change by altering function at many different levels in the nervous system from the subcellular to the network level. The output of a network depends on a complex interaction of structure and function at these different levels, including, for example, the types and number of different ion
636
Zebrafish
channels in the membranes of the various neurons in the circuit, the synaptic properties of the different connections in the network, and the exact pattern of wiring of the network. Not surprisingly, therefore, mutagenesis studies of zebrafish have revealed genes that disrupt organization at each of these different levels with profound behavioral consequences. A few examples, all isolated in the large Tu¨bingen screen, serve to illustrate some of the genetic effects that can alter more simple behaviors. This screen looked for larval fish in which the response to a touch was altered. These fell into many categories including those that did not respond at all and a whole series of animals that had aberrant movements subsequent to the touch. Unresponsive animals included those with major deficits in sensory neurons relaying information about the touch as well as in the motoneurons and muscle that are critical for producing the movement. In mutants such as macho, the excitability in sensory neurons was altered in a way that blocked their ability to produce action potentials and hence the touch could not trigger a movement. On the motor output side, a whole series of mutants with problems in the muscle or in the connection between nerve and muscle led to an inability to move in response to the touch. These included mutants such as relaxed which was a deficit in the dihydropyridine receptor that disrupted the calcium release in the muscle necessary for contraction. Others, such as sofa potato, were mutants of the acetylcholine receptors on the muscle, which rendered the muscle unresponsive to neurotransmitter released by the motoneurons. Many of the more interesting mutants led to altered movement patterns rather than to no movement at all. Disruption of the protein rapsyn that anchors acetylchoine receptors at the neuromuscular junction led to fish that could response to the stimulus, but could not sustain the resulting swimming movements. This mimics human disorders of muscle weakness, one of which involves a mutation similar to that identified in zebrafish. A whole class of mutants, called the accordion class, disrupts the ability of the fish to swim with the normal alternating bends from one side to the other; instead, muscle contractions overlap on the two sides in the mutants leading to an accordion like shortening of the body of the fish. Some of these involve slowed muscle contractions that lead to overlap in activity on the two sides of the body; they also include mutants with alterations in the central nervous system that reveal contributions of molecules to proper circuit function. One of these is the mutant bandoneon, which is a mutation in a beta subunit of a glycine receptor. This disrupts inhibitory synapses in the nervous system, including those that usually block activity in motoneurons on one side when the other side is active. The result is bilateral muscle activity. Mutants can also lead to a rewiring of the neural circuits by altered axonal projections of the neurons or alternatively, by changes in the numbers of particular cell types.
A mutation called space cadet produces a whole series of repeated escape-like bends to one side after a single touch. The evidence suggests that this is a consequence of missing pathways in the brain that cross from one side to the other and regulate the activity of neurons responsible for producing normal escapes. A striking mutant called deadly seven may offer some insight into the evolution of networks because it is a mutant of a gene notch that is important for controlling cell number in the nervous system. The mutant animals have extra Mauthner neurons, the cells that are a critical component of escape or startle behaviors. Interestingly, these extra neurons are wired into the escape network and the fish can produce robust escape movements. This suggests that extra neurons added by genetic alterations can be incorporated into circuits via normal developmental mechanisms without a dramatic effect on the behavior. This makes it easier to understand how an escape or startle behavior produced by few neurons in early aquatic vertebrates might have evolved into a startle response produced by much larger numbers of neurons in tetrapods. These are just a few examples of the studied mutants, but many other mutant lines of zebrafish have been isolated, but not yet studied at the genetic or functional levels. The evidence so far documents how even the simplest motor behaviors can be disrupted in many ways by mutants that affect all levels of neuronal and network function. The work serves to highlight the critical interplay between levels of functional organization in the nervous system that is essential to generate a proper behavioral response. More Complex Behaviors Complex behaviors are much harder to tackle at the genetic and neuronal level, but are of considerable biological interest. Studies of learning, sleep, aggression, more complex visual-motor behaviors, reward systems in the brain, and even zebrafish models of neurological disorders such as schizophrenia are opening the lines of attack on more complex behaviors. Most of these investigations are still in early stages, although some mutants with deficits in these behaviors have been isolated. The challenge here is to examine how the mutations affect the nervous system to alter the behavior. Analyzing this at the neuronal level depends to a great extent on understanding how the complex behavior is produced normally and this is something that is very difficult when the behaviors involve interactions of networks in many parts of the brain and spinal cord.
The Future The power of the zebrafish model will assure that it remains at the forefront of genetic and neuronal studies of behavior into the future. There is no other vertebrate
Zebrafish
model with comparable genetic, optical, electrophysiological, and behavioral accessibility. The most modern optical tools for using light to activate and inactivate neurons play to the zebrafish strength and are only in their infancy. When they are combined with transgenic approaches to target neuronal classes in normal and mutant lines of fish, they promise to forge very compelling ties between the genetic, neuronal, and behavioral level. Although much of the focus has been on simple sensory or motor behaviors, there is increasing attention to more complex behaviors and this is likely to grow, given the tools in zebrafish may offer the best attack on these behaviors. The links between behavior studied in the laboratory and behavior in the wild are still weak, but foundational work in the natural biology of the animal will hopefully seed other studies of issues such as social hierarchies, territory defense, and mate selection in this model, in which they may eventually be attacked at the genetic and neuronal levels. See also: Development, Evolution and Behavior; Fish Migration; Fish Social Learning; Genes and Genomic Searches; Nervous System: Evolution in Relation to Behavior; Neurobiology, Endocrinology and Behavior; Threespine Stickleback; Vocal–Acoustic Communication in Fishes: Neuroethology.
Further Reading Brockerhoff SE, Hurley JB, Janssen-Bienhold U, Neuhauss SC, Driever W, and Dowling JE (1995) A behavioral screen for isolating zebrafish mutants with visual system defects. Proceedings of the National
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Academy of Sciences of the United States of America 92: 10545–10549. Cui WW, Low SE, Hirata H, et al. (2005) The zebrafish shocked gene encodes a glycine transporter and is essential for the function of early neural circuits in the CNS. The Journal of Neuroscience 25: 6610–6620. Fetcho JR, Higashijima S, and McLean DL (2008) Zebrafish and motor control over the last decade. Brain Research Reviews 57: 86–93. Fetcho JR and Liu KS (1998) Zebrafish as a model system for studying neuronal circuits and behavior. Annals of the New York Academy of Sciences 860: 333–345. Granato M, van Eeden FJM, Schach U, et al. (1996) Genes controlling and mediating locomotion behavior in the zebrafish embryo and larva. Development 123: 399–413. Higashijima S (2008) Transgenic zebrafish expressing fluorescent proteins in central nervous system neurons. Development, Growth & Differentiation 50: 407–413. Liu KS and Fetcho JR (1999) Laser ablations reveal functional relationships of segmental hindbrain neurons in zebrafish. Neuron 23: 325–335. Neuhauss SC (2003) Behavioral genetic approaches to visual system development and function in zebrafish. Journal of Neurobiology 54: 148–160. Nicolson T (2005) The genetics of hearing and balance in zebrafish. Annual Review of Genetics 39: 9–22. O’Malley DM, Kao YH, and Fetcho JR (1996) Imaging the functional organization of zebrafish hindbrain segments during escape behaviors. Neuron 17: 1145–1155. Ono F, Shcherbatko A, Higashijima S, Mandel G, and Brehm P (2002) The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function. Journal of Neuroscience 22: 6491–6498. Orger MB, Gahtan E, Muto A, Page-McCaw P, Smear MC, and Baier H (2004) Behavioral screening assays in zebrafish. Methods in Cell Biology 77: 53–68. Spence R, Gerlach G, Lawrence C, and Smith C (2008) The behaviour and ecology of the zebrafish, Danio rerio. Biological Reviews of the Cambridge Philosophical Society 83: 13–34. Streisinger G, Walker C, Dower N, Knauber D, and Singer F (1981) Production of clones of homozygous diploid zebrafish (Brachidanio rerio). Nature 291: 293–296. Westerfield M (1995) The Zebrafish Book, 3rd edn. Eugene, OR: University of Oregon Press.
Introduction The zebrafish has only recently emerged as a key model for studies of the neuronal and genetic basis of behavior. George Streisinger’s pioneering studies at the University of Oregon established the zebrafish as a genetic model. Although he envisioned the animal as a model for using genetic tools to dissect behavior, for a long time the focus was on using zebrafish for studies of neuronal development. The initial advantages of this animal were its availability (it is a common pet store fish), its transparency, and the abundance of its eggs and larvae (which allowed easy imaging of neuronal development in the live animal), a somewhat short – 3 month – generation time, and some important mutant lines obtained by exposing the fish to mutagenic chemicals. These advantages led to some remarkable developmental/genetic studies by the Oregon group that put the zebrafish on the map as a model organism. Although there were scattered early behavioral studies of zebrafish, two developments growing out of the work in Oregon catalyzed the expansion of zebrafish as a model for behavioral studies. The first was a large-scale screen for mutants done in Tu¨bingen by the Nusslein-Vollhard group. While the focus of this screen was largely developmental and structural, one portion, led by Michael Granato, focused on animals with movement deficits. The isolation of these mutants catalyzed studies directed toward finding the disrupted genes and revealing how they affected patterns of movement. In parallel, the advantages of the transparency of the larval fish for functional studies became clear with work showing that one could load fluorescent calcium indicators into neurons to literally watch active nerve cells light up during behavior in the intact animal. This was the first imaging of neuronal activity with single-cell resolution in an intact vertebrate and raised the possibility of exploring activity patterns in vivo in both normal and mutant lines of fish. The combination of powerful tools at both the genetic and neuronal circuit level made zebrafish unique among the vertebrate models and set the stage for further behavioral studies. While much of the focus on zebrafish behavior has been in laboratory studies, recent work has begun to reveal more about their natural history and ecology. They are members of the Cyprinidae and are found in the flood plains of the Indian subcontinent, where they typically occur in shallow, slow-moving water with visibility 30 cm deep in areas with vegetation, little shade, and a silty bottom. The temperatures over the natural
range of the fish can vary from 6 to 38 C, although they are typically maintained at 28.5 C in the laboratory. The adults are omnivorous, with their food usually consisting of insects and zooplankton. Likely predators include other fish and birds. The fish typically spawn at first light in the wild, as in the lab, with groups of males chasing a female and the eggs becoming scattered upon release. There is some evidence that males protect sites containing gravel substrates that are preferred by females. Such sites may protect eggs from predation and provide them with higher oxygenation. While the studies upon which these observations of the natural biology of the animal are based are increasing, there is still much about features of their biology, such as intra and interspecific interactions, feeding, and habitat choice, that remain to be explored.
Behaviors of Zebrafish Zebrafish show the broad range of behaviors typical of other vertebrates. These extend from more simple sensorymotor reflex responses such as escape behaviors to complex social behaviors such as shoaling, aggression, and mate choice. A major focus of study has been to identify genes that build the primary sensory systems used to assess and respond to the external world including the visual system, the auditory system, the lateral line, and the somatosensory systems. On these fronts, the model has revealed unknown components of the hair cell apparatus for detecting movement in the lateral line and in the ear, as well as genes that are involved in setting up the neuronal and biochemical networks for appropriate visual responses. On the motor output side, contributions of genes to the production of swimming and escape behaviors have been revealed, along with the neuronal organization of these responses. Most of this behavioral work on the zebrafish model has involved laboratory studies of the genetic and neuronal substrates of relatively simple behaviors or parts of behaviors. There is less work dealing with natural variation and behavior in the wild, although this is beginning as well. Similarly, more complex mating and social behaviors have received some attention on the behavioral side, but are only beginning to move to the genetic and neuronal levels. Nonetheless, understanding the genetic and neuronal substrates of natural variation and complex behaviors is impossible without an understanding of the way in which core elements of particular behaviors are built at
633
634
Zebrafish
the genetic and neuronal levels, and there are now very powerful tools that allow attacks on behavior in zebrafish from the genetic through to the neuron and network levels. These are best considered in the light of what is required to achieve an adequate understanding of the proximate mechanisms by which genes and neurons influence the generation of a particular behavior because this is the arena where zebrafish may have the most to contribute as a model.
The Path from Genes to Behavior Genetic Perturbations The influence of genes on behavior is typically revealed by exploring the behavioral effects of perturbation of the activity of those genes. In zebrafish, there are several avenues for such perturbations. The most common is mutagenesis, done via chemical application to adult fish or via viral insertion into the genome to disrupt random genes and screen for behavioral changes. This is especially useful for revealing unknown genes that affect behaviors, but is not trivial as it typically involves large-scale raising of animals to isolate many mutants, sometimes involving many thousands of tanks of fish. A more directed approach uses morpholinos, modified oligonuceotides designed to bind to specific endogenous RNA sequences and disrupt their expression. These are targeted based upon sequence information, so the approach can test possible contributions to behavior of known genes, but cannot reveal novel ones. In addition to these tools, it is now easy to introduce exogenous genes into zebrafish to produce transgenic animals. This can be useful to express particular genes at higher levels by adding an additional copy under a known, possibly stronger than normal, promoter. This approach reveals the consequences of overexpression of a gene. What is currently missing in zebrafish, however, is the ability to remove or replace the endogenous genes by knockouts – a very powerful approach in other standard models for behavioral studies such as mice. Bridging the Gap Between Genes and Behavior The challenge in exploring the influence of genes on behavior is not so much in finding genes that affect behavior, as there are plenty of those in many species, but rather in bridging the gap between the genes and the behavior to reveal how the genes affect neurons and networks and in turn alter behavior. Some of the features of the zebrafish model make it particularly powerful and perhaps the best vertebrate model for bridging the gene to behavior gap. Moving from genes to behavior involves some critical considerations that include finding in which tissues or organs the gene is expressed and what alterations in
neuronal wiring or activity patterns occur following disruption of the gene as, in the end, these changes in the nervous system will alter behavior. Disrupting gene expression or creating overexpression may also cause changes in effector organs such as muscles or in neural connections with muscles, but some of the hardest challenges have been revealing the impact of genetic disruptions on the neuron or network level. The ability to make transgenic zebrafish and the transparency of the larval animal simplifies forging links between genes and neuronal structure and function. By inserting a fluorescent protein under control of the promoter for the gene of interest, the neurons expressing both the fluorescent protein and the protein of interest can be viewed in the intact, living larval fish. This allows direct visualization of which neurons express the gene. Observations of neurons treated in this way in mutant animals or those treated with morpholinos allow detection of changes in neuronal structure related to disruption of the gene of interest. Because disruptions might also occur in cells not expressing the gene, it is important to explore other aspects of neuronal organization as well. There are an increasing number of transgenic lines of zebrafish with different subsets of neurons labeled with different colored fluorescent proteins. These include lines with inhibitory and excitatory nerve cells labeled in different colors to, in effect, provide a color-coded nervous system. By looking at the effects of mutations or morpholinos in these lines, one can better reveal disruptions of neurons and neuronal patterning that might underlie behavioral changes. An additional advantage of zebra fish is the ability to easily examine the activity of labeled neurons, because genetic disruptions of behavior are usually linked to changes in neuronal activity patterns that can result from a mis-wired circuit, changes in the electrical properties of the neurons, or changes in the synaptic connections between the nerve cells. Every modern physiological tool for studying neuronal activity is accessible in the zebrafish model. Conventional approaches to directly recording the electrical activity of individual neurons with glass microelectrodes (such as patch clamp recording) are possible. More unique to zebrafish, the transparency of the larvae allows the imaging of neuronal activity anywhere in the animal. This has been accomplished with fluorescent indicators that sense calcium levels and change their fluorescence upon binding calcium. By loading neurons with such indicators in live fish, the active neurons literally light up during behavior. The availability of genetically encoded calcium indicators has allowed the generation of transgenic lines with built-in optical indicators of neuronal activity, first done among vertebrates in zebrafish. The methods for monitoring activity can be used in transgenic lines with particular cell types labeled, so the function of different subsets of neurons can be examined. These tools
Zebrafish
allow exploration of neuronal activity during behavior at the single cell and neuronal population levels. They are critical to defining both the normal networks underlying behavior as well as the consequences of genetic perturbations that alter behavior. Linking Neurons to Behavior One of the major challenges in studies of the neuronal basis of behavior is causally linking neurons or neuronal classes to particular behaviors. The approach to revealing these links is much the same as that for linking genes to behavior. The activity of the neurons must be perturbed in some way and the consequences for behavior studied. Zebrafish offer spectacular opportunities in this regard because of recently developed optical tools that play to the combination of genetics and transparency that are the strengths of the zebrafish model. Early perturbation studies used lasers focused on nerve cells in the intact animal to kill particular neurons and explore the behavioral consequences. Such removal is useful, but irreversible. Recently, genetically encoded proteins controlled by light were isolated that allow the electrical activity in nerve cells to be turned off or on by shining light on them. These tools take advantage of light activated ion pumps or channels that can affect the electrical activity of neurons. The methods work in zebrafish, and the ability to introduce genes into the fish and focus light anywhere in the nervous system will allow reversible inactivation or activation of different neuron classes to test their contribution to behavior. One can even imagine flashing neurons to turn them on in different patterns to essentially play activity patterns into the nervous system to examine the behavioral consequences of different patterns. The power of these approaches, which are best applied in a transparent animal, is likely to make the zebrafish model even more important in studies of the neuronal basis of behavior.
Examples of the Application of Optical and Genetic Approaches Neuronal Activity During Behavior The utility of zebrafish for behavioral work is best illustrated by examples from many contributions that take advantage of the special strengths of model. Some of the initial work taking advantage of the transparency for functional studies set out to reveal the patterns of neuronal activity in the hindbrain that underlie the escape response – a fast turning movement that fish use to avoid predators. There was good evidence that a set of nerve cells with axons projecting into the spinal cord, including the well-studied Mauthner neuron and other similar, serially repeated neurons in different parts of the
635
hindbrain, were key to generating the escape response, but their activity patterns during behavior were largely unknown. By labeling the neurons with fluorescent calcium indicators, it was possible to reveal differences in which neurons were active, depending on the location of a touch stimulus used to elicit an escape, because different neurons lit up during different escape stimuli. This approach linked patterns of activity in the hindbrain to different forms of a behavior and provided insight into the functional organization of a part of the brain critical to movement. Test of Neuronal Contributions to Behavior by Optical Perturbation The initial tests of neuronal contributions to behavior in zebrafish came from studies in which the contribution of hindbrain neurons active in different forms of escape was tested by laser ablation. This work confirmed in studies ablating individual cells that the different cells in the set of hindbrain neurons including the Mauthner neuron and others like it indeed made different contributions to the escape behavior. Killing particular cells could affect some forms of the escape response and not others, confirming the view that even a seemingly simple behavior can have subtle variations that can only be revealed with studies of an accessible model system. More recent work combined genetic approaches with laser ablations to test the contribution of neurons in spinal cord to movement. This work took advantage of a socalled enhancer trap line in which an insertion of a fluorescent protein into the genome led to the labeling of a very discrete set of spinal interneurons. These inhibitory cells had axons that crossed from one side of the body to the other and were thought to allow a forceful escape bend by blocking activation of muscle on the opposite side of the bend. Removing the neurons, which were easily targeted with a laser in the live animals because they were fluorescently labeled, led to a bilateral muscle contraction and a seriously disrupted escape bend. This directly confirmed the critical role of a class of neuron in a motor behavior. While these two examples focus on laser removal of neurons, the expectation is that future work will use reversible approaches to inactivating neurons with light to allow even more compelling studies of links of neurons to behavior. Genes Affecting Behavior A genetic change can produce a behavioral change by altering function at many different levels in the nervous system from the subcellular to the network level. The output of a network depends on a complex interaction of structure and function at these different levels, including, for example, the types and number of different ion
636
Zebrafish
channels in the membranes of the various neurons in the circuit, the synaptic properties of the different connections in the network, and the exact pattern of wiring of the network. Not surprisingly, therefore, mutagenesis studies of zebrafish have revealed genes that disrupt organization at each of these different levels with profound behavioral consequences. A few examples, all isolated in the large Tu¨bingen screen, serve to illustrate some of the genetic effects that can alter more simple behaviors. This screen looked for larval fish in which the response to a touch was altered. These fell into many categories including those that did not respond at all and a whole series of animals that had aberrant movements subsequent to the touch. Unresponsive animals included those with major deficits in sensory neurons relaying information about the touch as well as in the motoneurons and muscle that are critical for producing the movement. In mutants such as macho, the excitability in sensory neurons was altered in a way that blocked their ability to produce action potentials and hence the touch could not trigger a movement. On the motor output side, a whole series of mutants with problems in the muscle or in the connection between nerve and muscle led to an inability to move in response to the touch. These included mutants such as relaxed which was a deficit in the dihydropyridine receptor that disrupted the calcium release in the muscle necessary for contraction. Others, such as sofa potato, were mutants of the acetylcholine receptors on the muscle, which rendered the muscle unresponsive to neurotransmitter released by the motoneurons. Many of the more interesting mutants led to altered movement patterns rather than to no movement at all. Disruption of the protein rapsyn that anchors acetylchoine receptors at the neuromuscular junction led to fish that could response to the stimulus, but could not sustain the resulting swimming movements. This mimics human disorders of muscle weakness, one of which involves a mutation similar to that identified in zebrafish. A whole class of mutants, called the accordion class, disrupts the ability of the fish to swim with the normal alternating bends from one side to the other; instead, muscle contractions overlap on the two sides in the mutants leading to an accordion like shortening of the body of the fish. Some of these involve slowed muscle contractions that lead to overlap in activity on the two sides of the body; they also include mutants with alterations in the central nervous system that reveal contributions of molecules to proper circuit function. One of these is the mutant bandoneon, which is a mutation in a beta subunit of a glycine receptor. This disrupts inhibitory synapses in the nervous system, including those that usually block activity in motoneurons on one side when the other side is active. The result is bilateral muscle activity. Mutants can also lead to a rewiring of the neural circuits by altered axonal projections of the neurons or alternatively, by changes in the numbers of particular cell types.
A mutation called space cadet produces a whole series of repeated escape-like bends to one side after a single touch. The evidence suggests that this is a consequence of missing pathways in the brain that cross from one side to the other and regulate the activity of neurons responsible for producing normal escapes. A striking mutant called deadly seven may offer some insight into the evolution of networks because it is a mutant of a gene notch that is important for controlling cell number in the nervous system. The mutant animals have extra Mauthner neurons, the cells that are a critical component of escape or startle behaviors. Interestingly, these extra neurons are wired into the escape network and the fish can produce robust escape movements. This suggests that extra neurons added by genetic alterations can be incorporated into circuits via normal developmental mechanisms without a dramatic effect on the behavior. This makes it easier to understand how an escape or startle behavior produced by few neurons in early aquatic vertebrates might have evolved into a startle response produced by much larger numbers of neurons in tetrapods. These are just a few examples of the studied mutants, but many other mutant lines of zebrafish have been isolated, but not yet studied at the genetic or functional levels. The evidence so far documents how even the simplest motor behaviors can be disrupted in many ways by mutants that affect all levels of neuronal and network function. The work serves to highlight the critical interplay between levels of functional organization in the nervous system that is essential to generate a proper behavioral response. More Complex Behaviors Complex behaviors are much harder to tackle at the genetic and neuronal level, but are of considerable biological interest. Studies of learning, sleep, aggression, more complex visual-motor behaviors, reward systems in the brain, and even zebrafish models of neurological disorders such as schizophrenia are opening the lines of attack on more complex behaviors. Most of these investigations are still in early stages, although some mutants with deficits in these behaviors have been isolated. The challenge here is to examine how the mutations affect the nervous system to alter the behavior. Analyzing this at the neuronal level depends to a great extent on understanding how the complex behavior is produced normally and this is something that is very difficult when the behaviors involve interactions of networks in many parts of the brain and spinal cord.
The Future The power of the zebrafish model will assure that it remains at the forefront of genetic and neuronal studies of behavior into the future. There is no other vertebrate
Zebrafish
model with comparable genetic, optical, electrophysiological, and behavioral accessibility. The most modern optical tools for using light to activate and inactivate neurons play to the zebrafish strength and are only in their infancy. When they are combined with transgenic approaches to target neuronal classes in normal and mutant lines of fish, they promise to forge very compelling ties between the genetic, neuronal, and behavioral level. Although much of the focus has been on simple sensory or motor behaviors, there is increasing attention to more complex behaviors and this is likely to grow, given the tools in zebrafish may offer the best attack on these behaviors. The links between behavior studied in the laboratory and behavior in the wild are still weak, but foundational work in the natural biology of the animal will hopefully seed other studies of issues such as social hierarchies, territory defense, and mate selection in this model, in which they may eventually be attacked at the genetic and neuronal levels. See also: Development, Evolution and Behavior; Fish Migration; Fish Social Learning; Genes and Genomic Searches; Nervous System: Evolution in Relation to Behavior; Neurobiology, Endocrinology and Behavior; Threespine Stickleback; Vocal–Acoustic Communication in Fishes: Neuroethology.
Further Reading Brockerhoff SE, Hurley JB, Janssen-Bienhold U, Neuhauss SC, Driever W, and Dowling JE (1995) A behavioral screen for isolating zebrafish mutants with visual system defects. Proceedings of the National
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Academy of Sciences of the United States of America 92: 10545–10549. Cui WW, Low SE, Hirata H, et al. (2005) The zebrafish shocked gene encodes a glycine transporter and is essential for the function of early neural circuits in the CNS. The Journal of Neuroscience 25: 6610–6620. Fetcho JR, Higashijima S, and McLean DL (2008) Zebrafish and motor control over the last decade. Brain Research Reviews 57: 86–93. Fetcho JR and Liu KS (1998) Zebrafish as a model system for studying neuronal circuits and behavior. Annals of the New York Academy of Sciences 860: 333–345. Granato M, van Eeden FJM, Schach U, et al. (1996) Genes controlling and mediating locomotion behavior in the zebrafish embryo and larva. Development 123: 399–413. Higashijima S (2008) Transgenic zebrafish expressing fluorescent proteins in central nervous system neurons. Development, Growth & Differentiation 50: 407–413. Liu KS and Fetcho JR (1999) Laser ablations reveal functional relationships of segmental hindbrain neurons in zebrafish. Neuron 23: 325–335. Neuhauss SC (2003) Behavioral genetic approaches to visual system development and function in zebrafish. Journal of Neurobiology 54: 148–160. Nicolson T (2005) The genetics of hearing and balance in zebrafish. Annual Review of Genetics 39: 9–22. O’Malley DM, Kao YH, and Fetcho JR (1996) Imaging the functional organization of zebrafish hindbrain segments during escape behaviors. Neuron 17: 1145–1155. Ono F, Shcherbatko A, Higashijima S, Mandel G, and Brehm P (2002) The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function. Journal of Neuroscience 22: 6491–6498. Orger MB, Gahtan E, Muto A, Page-McCaw P, Smear MC, and Baier H (2004) Behavioral screening assays in zebrafish. Methods in Cell Biology 77: 53–68. Spence R, Gerlach G, Lawrence C, and Smith C (2008) The behaviour and ecology of the zebrafish, Danio rerio. Biological Reviews of the Cambridge Philosophical Society 83: 13–34. Streisinger G, Walker C, Dower N, Knauber D, and Singer F (1981) Production of clones of homozygous diploid zebrafish (Brachidanio rerio). Nature 291: 293–296. Westerfield M (1995) The Zebrafish Book, 3rd edn. Eugene, OR: University of Oregon Press.