There have been several studies that have characterized schizophrenia-related genes in zebrafish. For example, the connection between Akt signaling, dopaminergic neurotransmission, and schizophrenia has been investigated. Cheng et al. (2013) have used zebrafish to study *rgs4*, a regulator of G-protein signaling family that is expressed in the developing nervous system. Loss of gene function causes both neurite outgrowth defects in the hindbrain and spinal cord and a reduction of motility. Activation of Akt signaling can rescue the outgrowth phenotype in the spinal cord but not hindbrain, indicating that Akt signaling is required for some *rgs4*-mediated axon formation (Cheng et al., 2013). Souza et al. (2011) have further examined the connection between Akt signaling and dopaminergic neurotransmission in the developing brain. Since alterations in Akt signaling have also been linked to schizophrenia, this strengthens the suggestion that altered dopaminergic signaling during development might underlie the disease. Other human disease-causing genes have been studied as well. Knock-down of the synaptically expressed schizophrenia-susceptibility gene *kinesin17* (*kif17*) causes a severe phenotype in zebrafish embryos including stunted development and a curly tail phenotype suggesting that *kif17* is active during embryonic development (Tarabeux et al., 2010). Finally, treatment of zebrafish with the NMDA receptor antagonist MK-801 causes several behavioral alterations including changes to social interaction, hyperactivity, and amnesia, which have been interpreted as being schizophreniform (schizophrenia-like; Chen et al., 2010; Seibt et al., 2010, 2011; Echevarria et al., 2011) and are similar to the behavioral changes elicited by these psychotics in rodents.

In summary, although it is not possible to model some of the more complex psychological symptoms of schizophrenia in fish, several recent studies have given insights into some of the basic processes underlying this disease.

## **ZEBRAFISH MODELS OF AUTISM SPECTRUM DISORDER**

Autism spectrum disorder encompasses a range of psychiatric diseases including autism (the severest form of ASD), Rett's syndrome, and Asperger's syndrome. The symptoms of ASD are highly variable between patients making it difficult uncover the genetic and neurobiological changes that underlie these disorders. Broadly speaking, the main symptoms of ASD include alterations in social behavior, repetitive behavior, and language development that appear before 3 years of age (Lord et al., 2000). There is a heritability estimate of between 70 and 80% for ASD (Bailey et al., 2005; Rosenberg et al., 2009). However, the number of people thought to suffer from ASD is increasing over time which might be due to a shift in the diagnostic criteria used (Lord, 2010). Although it is not possible to study complex behaviors such as language development in fish, a few recent studies have provided information about thefunction of ASD-linked genes during neural development.

Both common and rare (*de novo*) genetic variants, acting in combination with environmental influences, are associated with ASD. ASD-linked polymorphisms are predominantly found in genes that regulate synapse development, cell proliferation, neural migration, and neural projection. Rare mutations seem to account for around 10% of cases of ASD (Geschwind, 2008). It has been suggested that there may be a different genetic basis for simplex families (where only one child is affected and rare mutations are common) and multiplex families (where multiple genetic variants lead to ASD expression in several family members; Geschwind, 2008). For example, there is a higher frequency of CNVs in ASD families with only one affected child compared to those with two or more ASD children (Toro et al., 2010). Interestingly, there is also some data suggesting that rare and common variants interact to trigger ASD symptoms (Ben-David and Shifman, 2012). Genes which have been linked toASD include the Fragile X gene *FRAGILE X MENTAL RETARDATION 1* (*FMR1*), the GABAA β 3 subunit gene *GABRB3* as well as SHANK3, *TSC1*, *NEUROLIGIN3* and *NEUROLIGIN4*, *PTEN* and *CNTNAP2* (Cook and Scherer, 2008; Geschwind, 2008, 2011; Lord, 2010) amongst others. The neurobiology of ASD is not well understood, but most likely includes brain areas which are needed for the control of social behavior and language. For example, altered connections between the frontal lobe (the orbitofrontal cortex) and temporal lobe (including the superior temporal gyrus and temporal polar cortex) may mediate some symptoms of the disease (Geschwind, 2011). Other important brain areas include the cerebellum, brainstem, and limbic areas including the hippocampus, amygdala, septal nuclei, and the anterior cingulate cortex (Lord et al., 2000). Finally, autism has also been related to megalencephaly, an overall increase in brain size in some patients.

Since the symptoms of ASD first occur during neural development it is possible to study the early function of ASD-related genes in this species (Tropepe and Sive, 2003). There are only a few research articles reporting the use of zebrafish in this type of research. In humans, a CNV at 16p11.2 has been associated with susceptibility to suffer from several disorders, including ASD, epilepsy, autism, and schizophrenia. The 16p11.2 CNV has been studied in zebrafish by two groups (Blaker-Lee et al., 2012; Golzio et al., 2012). In a landmark study, Golzio et al. (2012) analyzed 24 of the genes contained within this CNV and identified one, *KCDT13*, as most likely being causative for the disease. Overexpression of *kcdt13* (by mRNA injection) led to microcephaly, whereas gene-specific morpholino knock-down caused an increase in head size, likely due to alteration of the cell cycle in both cases. This study provides a neat demonstration of the use of zebrafish to identify disease-causing variants within large genomic regions. Gauthier et al. (2010) have used morpholinos to knockdown *SHANK3*, a gene linked to schizophrenia and ASD in human patients. *shank3* morphant zebrafish show decreased swimming after being touched, a phenotype which can be rescued with a wild-type (but not a mutant) version of the corresponding rat gene. Therefore, this study validated alterations to *shank3* as being

"fncir-07-00079" — 2013/4/25 — 12:54 — page 6 — #6

important in the control of behavior, with potential implications for both schizophrenia and ASD.

## **ZEBRAFISH MODELS OF MENTAL RETARDATION**

Mental retardation constitutes a significant impairment of cognitive function that can be symptomatic of a number of underlying neural defects. In this review, I have chosen to focus on XLMR (also called Fragile X syndrome, FXS) a common inheritedform of mental retardation which affects around 1 in 4000 people. However, zebrafish have also been used to examine other types of mental retardation (for recent studies, refer to Komoike et al., 2010; Song et al., 2010; Brockschmidt et al., 2011; Friedrich et al., 2012; Veleri et al., 2012; Aspatwar et al., 2013). The symptoms of FXS include mental retardation, epilepsy, autistic-like behavior, attention deficits, macroorchidism, and mild craniofacial defects which have been linked to the maturation of dendritic spines during development.

Similar to schizophrenia and ASD, it is likely to be impossible to model the cognitive symptoms of mental retardation in fish. However, several zebrafish groups have analyzed XLMR-linked genes and provided information about their role during development. For example, XLMR is most often caused by mutations in the *FMR1* gene, which contains a variable CGG trinucleotide repeat sequence in its 5- UTR (Verkerk et al., 1991). Once this repeat passes 200 copies, the gene becomes hypermethylated and loss of function occurs. Interestingly, this methylation appears to be mediated by a microRNA which is present in the 3- UTR of *FMR1*, an observation which has been experimentally confirmed in zebrafish (Lin et al., 2006). Injection of a construct targeting the anti-fmr1 miRNA causes abnormal neural morphology and an increase in the size of the head (Lin et al., 2006).

Studies of the zebrafish homolog of the *FMR1* gene have given equivocal results. Intriguingly, different phenotypes were observed when comparing morpholino-mediated knock-down and a stable *fmr1* mutant line. Morphant larvae show craniofacial defects, hydrocephaly, and pericardial edema as well as abnormal branching of the trigeminal ganglion and lateral longitudinal fasciculus (Tucker et al., 2006). However, in contrast to this, *fmr1* mutant larvae are adult viable and show no detectable morphological differences (Den Broeder et al., 2009). Thus, it is clear that further studies are needed in order to fully understand the function of this gene during development. Qi et al. (2010) studied the XLMR gene *phf8* during zebrafish development. *phf8* is a mono-methyl histone H4 lysine 20 demethylase that is expressed in the developing head and jaw of zebrafish at 1 day. Loss of gene function causes severe disruption to development of these structures, most likely caused by increases in apoptosis (Qi et al., 2010). Histone methylation has also been linked to XLMR in a study of the *smcx/jarid1c* gene, a H3K4 demethylase (Iwase et al., 2007). Loss of *smcx* function during development leads to increased cell death and a smaller brain as well as a reduced number of dendrites. Together, both of these studies suggest that epigenetic events might play an important role in the etiology of XLMR. Another XLMR-linked gene, *rbmx*, shows strong expression in the anterior developing embryo including the brain (Tsend-Ayush et al., 2005). Knock-down of *rmbx* leads to a reduction in body size, pericardial edema, hydrocephaly, and reduced motility (Tsend-Ayush et al., 2005).  Knock-down of one of the homologs, *ilrapl1b*, inhibits presynaptic differentiation during development (Yoshida and Mishina, 2008).

There are several learning and choice discrimination tests that have been developed for adult zebrafish which could be used to test possible cognitive functions of mental retardation-associated genes. For example, Brennan and colleagues have developed a 3-choice serial reaction time task (3CSRTT) that can be used to measure impulsivity (Parker et al., 2012). The 3CSRTT is measured in a tank that has a green light-emitting diode (LED) on one side and three yellow LEDs in separate compartments on the other. Following illumination of the green LED, adult zebrafish are taught to only enter the compartment where the yellow LED is switched on. The correct execution of this behavior is reinforced with a food reward. Following a training period in which the fish learns to associate the yellow light with a reward the 3CSRTT can begin. The green stimulus LED is first activated and is then followed by a 10-s intertrial interval (ITI). Following this pause, one of the yellow LEDs is lit and the fish is rewarded with food upon entering the correct compartment. However, entry into any compartment before the end of the ITI, perhaps indicative of impulsivity, will result in a punishment (a 10-s time-out with no food). Entry into an incorrect compartment on the other side (i.e., one in which the yellow LED is not illuminated) will also trigger the punishment. Other tests of learning and memory, including associative-, avoidance-, and spatial memory have also been established (reviewed in Norton and Bally-Cuif, 2010). Although these tests appear to be promising in to study zebrafish cognition, they have mostly only been tested on wild-type fish and so need further validation (for example, in fish lacking the function of a diseaselinked gene) before they can be proposed as retardation-linked phenotypes.

## **FUTURE DIRECTIONS FOR RESEARCH INTO PSYCHIATRIC DISORDERS USING ZEBRAFISH**

The functions of genes linked to psychiatric disorders are still relatively understudied in zebrafish. However, the power of zebrafish as a model organism to study neural development and behavior suggest that there are several areas in which the zebrafish has the potential to provide more information related to these diseases, as discussed below.

### **ENVIRONMENTAL CONTRIBUTIONS TO DISEASE ONSET**

Since zebrafish embryos develop outside of their mother, it is easy to manipulate their environment and so carry out G × E studies. Although it is not possible to model adverse psychosocial environments (such as parenting styles) in fish, other environmental factors including exposure to alcohol or nicotine could easily be studied. Ethanol exposure during development causes a range of phenotypes depending on the timing and concentration used, making it difficult to get an overview of the mechanism underlying the action of this drug, or how it might relate to alcohol-related changes to behavior in humans. At concentrations greater than 2%, ethanol causes gross body abnormalities including cardiac edema,

"fncir-07-00079" — 2013/4/25 — 12:54 — page 7 — #7

cyclopia, and dysmorphia (Blader and Strahle, 1998; Sylvain et al., 2010). Lower concentrations trigger behavioral alterations in the absence of obvious morphological changes. For example, transient exposure to ethanol can lead to either locomotor hyperactivity (Tal et al., 2012), a reduction of swimming coupled to defects in secondary motorneuron development and synaptogenesis (Sylvain et al., 2010, 2011) or reduced shoaling in adult fish (Fernandes and Gerlai, 2009; Buske and Gerlai, 2011). Embryos which are treated with nicotine during development tend to be smaller, with a permanent delay and disruption of secondary motorneuron development (Svoboda et al., 2002; Menelaou and Svoboda, 2009). Nicotine also causes a transient increase in swimming speed, followed by a long-lasting reduction in both startle response and swimming speed, a phenotype which is most likely mediated via activation of nAChRs (Svoboda et al., 2002; Eddins et al., 2010).

One of the difficulties of designing G×E studies, highlighted by the examples related to ethanol and nicotine, will be to mimic the situation in nature as closely as possible. Although environmental alterations may modify the severity of an obvious pre-existing phenotype, in some cases environmental influences would be expected to trigger disease-related symptoms in a genotype that initially looks normal. One way to address this issue would be to develop a zebrafish transgenic line that contains a humanized form of the disease-risk gene and then subject embryos to different environmental conditions (such as varying concentrations of ethanol or nicotine exposure) before measuring behavior. Although such an approach is not more technically difficult than starting with a measurable behavioral change, it would require researchers to have more confidence when developing the assay.

## **SCREENING FOR NOVEL DRUGS**

Zebrafish are a good model system for pharmacological studies, since compounds can be diluted in the embryo medium, and larvae are transparent meaning that internal organs can be visualized throughout development (Peterson et al., 2000). Furthermore, larvae are small, easy to generate in large numbers and easy to manipulate making them ideal for high-throughput work. However, since screens that use behavior as a read-out are difficult to design and implement efficiently (particularly if focusing on complex behaviors other than locomotion), it might be easier to carry out an initial pre-screen of a disease endophenotype. For example, zebrafish lacking the function of the ADHD-linked gene *lphn3.1* show a reduction and displacement of dopaminergic neurons in the PT of the diencephalon (Lange et al., 2012). Pre-screening libraries for compounds which can rescue this phenotype might speed up the screening process, with a subset of promising drugs then being re-tested to assess their behavioral function.

Two studies have combined analysis of underlying chemical structure and high-throughput screening of chemical libraries to look for alterations to the photomotor response, an embryonic motor response to a series of light flashes (Kokel et al., 2010; Laggner et al., 2012). This assay can be performed in a 96-well plate, and can be scaled up to analyze around 5000 novel chemicals every day. Rihel et al. (2010) uncovered the effect of drugs on locomotor activity across different sleep/wake cycles, providing novel

information about neuroactive drugs which can affect behavior. In this study, chemicals were categorized according to their "behavioral fingerprint," a combination of their chemical structure and their ability to modify aspects of behavior, thus permitting prediction of the function of novel chemicals.

A screen for novel anti-epileptic drugs was conducted in 2-dayold zebrafish embryos by Baxendale et al. (2012). Although not a developmental psychiatric disorder, this work further demonstrates the utility of zebrafish for drug identification. Exposure of embryos to the convulsant pentylenetetrazole (PTZ) upregulates expression of the immediate-early gene *fos* in the brain. The level of *fos* expression (assayed by *in situ* hybridization) was then used to screen a bioactive small-molecule library with a hit rate of 2.3% for anti-convulsive drugs (Baxendale et al., 2012). Using a similar approach, Baraban (2007) screened for novel zebrafish mutant lines which are resistant to the seizure-inducing properties of PTZ at 7 days post fertilization. Six novel mutant lines were identified, raising the possibility of uncovering novel genes with a seizure-protective function. The effects of the stimulants ethanol, amphetamine, and cocaine on larval swimming behavior have also been reported (Irons et al., 2010). Whilst not high-throughput, this study demonstrates the ease of screening motor behavior in 96-well plates, meaning that this paradigm could potentially be used to look at hyperactivity or startle, endophenotypes related to psychiatric disorders.

Although zebrafish constitute a power model system for drug screens, further experiments will be required before data can be translated to human patients. In a first step, the results will need to be verified in a second animal model – most likely a knockout mouse harboring a similar genetic lesion. Analysis of further disease models with and without drug treatment will provide information about selectivity of the chemical compound for one or more signaling pathways. A drug with a similar profile in both fish and mouse will represent a very promising target for clinical trials as long as no toxic effects are detected.

## **CONCLUSION**

A full understanding of the basis of psychiatric disorders requires the characterization of complex behaviors such as thought, feeling, and emotion, most likely through translational experiments in a combination of model organisms such as zebrafish and rodents. However, the suitability of zebrafish for developmental studies, and the genetic tools which are available to manipulate them suggest that the early development function of psychiatric disorder-linked genes can be studied in this model organism. Studies of genes linked to ADHD, schizophrenia, ASD, and mental retardation that have been performed in zebrafish have already demonstrated the validity of this approach. I look forward to future developments in this exciting field.

## **ACKNOWLEDGMENTS**

I am grateful to Marion Coolen, Merlin Lange, and Mike Jay for critically reading an earlier version of this manuscript. Many of the ideas and concepts presented in this review were formed during discussions with Laure Bally-Cuif and colleagues in Gifsur-Yvette. I am indebted to these people for their input and support.

"fncir-07-00079" — 2013/4/25 — 12:54 — page 8 — #8

## **REFERENCES**

hyperactivity disorder. *Arch. Gen. Psychiatry* 53, 607–616.

Generation and characterization of FMR1 knockout zebrafish. *PLoS ONE* 4:e7910. doi: 10.1371/journal.pone.0007910

"fncir-07-00079" — 2013/4/25 — 12:54 — page 9 — #9

novo mutation rate in individuals with schizophrenia. *Nat. Genet.* 43, 860–863.

"fncir-07-00079" — 2013/4/25 — 12:54 — page 10 — #10

gene and attention deficit hyperactivity disorder. *Psychiatr. Genet.* 15, 53–59.

mutations; one mutation, multiple disorders. *Trends Genet.* 25, 528–535.

"fncir-07-00079" — 2013/4/25 — 12:54 — page 11 — #11

	- Walsh, C. A. (1999). Genetic malformations of the human cerebral cortex. *Neuron* 23, 19–29.

chromosomal deletions and duplications in attention-deficit hyperactivity disorder: a genome-wide analysis. *Lancet* 376, 1401–1408.

A. L., et al. (2009). CNTNAP2 and NRXN1 are mutated in autosomalrecessive Pitt–Hopkins-like mental retardation and determine the level of a common synaptic protein in *Drosophila*. *Am. J. Hum. Genet.* 85, 655–666.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 24 January 2013; accepted: 09 April 2013; published online: 26 April 2013.*

*Citation: Norton WHJ (2013) Toward developmental models of psychiatric disorders in zebrafish. Front. Neural Circuits 7:79. doi: 10.3389/fncir.2013.00079*

*Copyright © 2013 Norton. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

"fncir-07-00079" — 2013/4/25 — 12:54 — page 12 — #12

## The role of zebrafish (Danio rerio) in dissecting the genetics and neural circuits of executive function

## *Matthew O. Parker, Alistair J. Brock, Robert T. Walton and Caroline H. Brennan\**

*School of Biological and Chemical Sciences, Queen Mary University of London, London, UK*

#### *Edited by:*

*Gonzalo G. De Polavieja, Instituto Cajal - Consejo Superior de Investigaciones Científicas, Spain*

#### *Reviewed by:*

*David Parker, Cambridge University, UK Akira Muto, National Institute of*

#### *Genetics, Japan \*Correspondence:*

*Caroline H. Brennan, School of Biological and Chemical Sciences, Queen Mary University of London, Mile End, London E1 4NS, UK. e-mail<EMAIL_ADDRESS>
Zebrafish have great potential to contribute to our understanding of behavioral genetics and thus to contribute to our understanding of the etiology of psychiatric disease. However, progress is dependent upon the rate at which behavioral assays addressing complex behavioral phenotypes are designed, reported and validated. Here we critically review existing behavioral assays with particular focus on the use of adult zebrafish to explore executive processes and phenotypes associated with human psychiatric disease. We outline the case for using zebrafish as models to study impulse control and attention, discussing the validity of applying extant rodent assays to zebrafish and evidence for the conservation of relevant neural circuits.

**Keywords: zebrafish, attention, impulsivity, behavioral flexibility, psychiatric disorder, neural circuits**

## **INTRODUCTION**

Gaining a better understanding of the etiology and pathogenesis of psychiatric disease is currently a priority area of research (Campbell, 2010). Advances in human neuroimaging and genetics are giving insight into the cellular regions and processes involved. However, partly because studies in humans must deal with genetic, diagnostic and etiological heterogeneity as well as environmental (cultural, societal) factors, and it is not possible to undertake molecular studies *in vivo*, progress remains slow (Burmeister et al., 2008). In order to address some of these concerns animal, primarily rodent, models targeting symptoms consistent with DSM-IV (APA, 2000) diagnoses of psychiatric disorder have been developed (Gould and Gottesman, 2006). Recently, with the establishment of zebrafish as a developmental genetic model with unparalleled utility for neural imaging, the potential of this genetically tractable vertebrate as a model in behavioral neuroscience has started to be realized (Levin and Cerutti, 2009; Norton and Bally-Cuif, 2010; Levin, 2011; Gerlai, 2012; Parker and Brennan, 2012).

In this paper, we review the current position with regards to the development and validation of zebrafish behavioral assays pertinent to human psychiatric disorder. We present an overview of neural pathways underlying key behaviors in rodents and the evidence for their conservation in fish. Finally we discuss prospects for the future: in particular, ways in which zebrafish can contribute to our understanding of cellular and molecular processes underlying psychiatric disease. Despite the numerous benefits of larval models and the progress that has been made in recent years (see Ahmad et al., 2012 for recent review), the utility of larvae to measure some of the subtle endophenotypes pertinent to vulnerability to psychiatric disorders may be limited. For example, although analysis of unconditioned or reflexive behaviors is clearly possible, it is unlikely that studying endophenotypes relative to cognitive or executive processes would be suitable owing to the immaturity of

the larval neural systems. Thus here, we focus on adult behavioral phenotypes.

## **THE BENEFITS OF DEVELOPING ZEBRAFISH MODELS OF BEHAVIORAL PHENOTYPES ASSOCIATED WITH PSYCHIATRIC DISEASE**

There are a great many practical benefits of using zebrafish as a model organism, e.g., their small size and low housing costs, their transparent, rapidly developing, ex-utero embryos, and their unsurpassed genetic tractability. However, in the age of technological advances in *in vivo* methodologies, such as optogenetics, enabling modulation of cell physiology and activity at the single cell level, their utility may soon be even greater. In this section, we discuss the potential for using zebrafish for behavioral assays pertinent to psychiatric disease.

There are inherent difficulties in comparing behaviors observed in non-primate species – particularly those associated with psychiatric disease – with those seen in humans (Brown and Bowman, 2002). One key issue is the lack of an expanded telencephalon and prefrontal cortex (PFC), the primary areas controlling executive functions commonly disrupted in psychiatric disease. There did exist a central dogma in neuroscience that cognitive processes have evolved in concert with the expansion of the telencephalon and lamination of the cortex, and therefore, animals without this expansion cannot perform these behaviors. However, there is now increasing evidence that smaller-brained vertebrates, which lack expanded telencephali, are capable of cognitive processing and even complex decision-making. For example, non-mammalian brains that do not have a laminar structure, such as nucleated bird brains, (a) show complex cognition, (b) have similar neural and neurochemical systems (especially dopamine(DA) and (c) display executive functions like mammals which are controlled by homologous brain structures (nidopallium instead of PFC; Jarvis et al., 2005). Thus it seems that several species may have faced

"fncir-07-00063" — 2013/4/5 — 12:30 — page 1 — #1

niche-specific selection pressures leading to evolution of comparable executive processes. This suggestion raises two questions regarding the use of zebrafish to explore molecular and cellular processes contributing to psychiatric disease: first, *do* fish show comparable behaviors? Second, if fish *can* perform executive tasks, have different regions or systems within their brains evolved to perform the tasks; or alternatively, have simple circuits (present in rudimentary form in common ancestors) evolved, albeit with topographical differences and different degrees of sophistication, to perform the same tasks, i.e., are the behavioral processes analogous or homologous? If the behaviors in fish, birds, and mammals share neurochemical pathways and show similar connectivity, it would suggest a common root and that the processes are homologous. In this event studies in fish, that do not model primate PFC executive function *per se,* can allow the extrapolation of common cell:cell interactions and physiological processes to give insight into molecules involved (in the human condition) despite differences in topography.

As the zebrafish is a uniquely tractable vertebrate genetic model species, assays of endophenotypes associated with psychiatric disease have been established and work to determine the neural pathways involved is underway. However, in the light of differences in structures, and possible differences in connectivity, there is a need for careful validation of the behavioral assays in zebrafish to establish their relevance to the human condition.

A good model of a human disease phenotype needs to demonstrate face-validity (the model *looks like* it is measuring the disease in question), construct validity (whether it *actually measures* what it claims to measure) and predictive power (Gould and Gottesman, 2006). As rodent models fulfill these criteria in many cases, zebrafish researchers have modified extant rodent assays, taking into account the specific behavioral system of the species. An example of this would be the novel tank test (Levin et al., 2007; Parker et al., 2012c). The novel tank test is an adaptation of the rodent open-field test of anxiety, developed with high face validity as it was designed with the zebrafish's natural tendency to dive to the bottom of a new environment in mind. Construct validity has been demonstrated using anxiolytic drugs; buspirone; and diazepam reduce the amount of time spent in the bottom of the tank in a dose-dependent manner (Bencan et al., 2009). For other behaviors, rapid progress is being made and in many instances construct validity has been similarly established by the use of pharmacological manipulation and genetic loss-of-function lines. In this section, we will briefly describe some assays used in rodent models of psychiatric disease, and how these have been adapted for, and validated in, zebrafish. Although a number of different behavioral phenotypes can be linked to psychiatric disease, such as anxiety, in this review we will concentrate on disorders relating to executive functioning rather than affective state, which has been reviewed extensively elsewhere (e.g., see Maximino et al., 2010).

## **BEHAVIORAL FLEXIBILITY**

Deficits in cognitive or behavioral flexibility are commonly reported in patients with a variety of psychiatric diagnoses, such as somatoform disorders (anorexia nervosa, bulimia nervosa: Button et al., 2007; Cardona et al., 2011), bipolar disorder (Balleine and Dickinson,1998), schizophrenia (Fernandez-Ruiz et al.,2001), substance abuse disorders (Collins et al., 2011) and obsessive compulsive disorder (OCD; Brembs, 2009). Behavioral flexibility is operationally defined as the ability to shift or adapt response strategy in the face of changing environmental contingencies (Ragozzino et al., 1999).

In animal models, behavioral flexibility may be measured by serial reversal of contingencies in two-choice discriminations, or by intra- and extra-dimensional (ED) set-shift tasks (see **Figure 1**; Ragozzino et al., 1999). Convergence of evidence from pharmacological (Saus et al., 2010), lesion (Hoopes, 1999) and genetic knockout (White et al., 2008) studies suggest a high degree of construct validity when compared with human task performance on tests of behavioral flexibility. Typically, the assay involves the

"fncir-07-00063" — 2013/4/5 — 12:30 — page 2 — #2

animal being trained to discriminate two stimuli (e.g., blue vs. green light), where in the first instance responses to the blue light are reinforced, and those to the green non-reinforced. Once the animal reaches a set criterion, the contingency is reversed, such that responses to the green light are now reinforced, and responses to the blue light non-reinforced. Subsequently, the colors can be changed (intra-dimensional [ID] shift) and reversed, or a third dimension can be introduced (e.g., shape; ED shift) and subsequently reversed. Many studies have demonstrated that rodents (Hoopes, 1999), primates (Doyle et al., 2006) and birds (Wilens et al., 2005) show a gradual improvement in their trials-to-criteria in this context.

Zebrafish are capable of reversal learning (Colwill et al., 2005), and recently they have been shown to follow a similar pattern of improvement over multiple reversals and ID shift as mammals. Parker et al. (2012a) trained adult zebrafish first on a simple color discrimination, once the fish had reached criterion (6 consecutive correct trials for 2 consecutive training sessions) the contingencies were reversed. Once the fish had reached criterion on the reversal condition, they were subjected to an ID shift, and subsequently contingency reversal. As has been shown in mammals, trials-to-criterion reduced during the course of the four training phases suggesting that the fish had formed an attentional-set, and could demonstrate flexibility in their learning with a changing environment. These findings were of particular interest, as they were the first to suggest that zebrafish were capable of behavioral flexibility in this context, complementing previous work in other fish species (e.g., goldfish; Woodward et al., 1971). Other, more ethological approaches to studying behavioral flexibility have also been used. Oliveira (2009), for example, observed that zebrafish adapt their social behavior dependent on outcomes of conflicts between conspecifics. These data raise the possibility of examining the cellular and molecular processes governing the operation of neural circuits involved in behavioral flexibility using zebrafish.

### **ATTENTION**

Attention can be described as *selective*, operationally defined as the ability to pick a target from an array of distracters (Desimone and Duncan, 1995), or *sustained*, operationally defined as the ability to detect the presence of a stimulus presented at various intervals over a prolonged period (Sarter et al., 2001). Deficits in sustained and selective attention are common features of a number of psychiatric disorders such as attention-deficit hyperactivity disorder (ADHD; Barkley, 1997), schizophrenia (Meshorer and Soreq, 2006), OCD (Kume et al., 2005), and substance abuse disorders (Tsutsui-Kimura et al., 2010). Both selective and sustained attention can be measured in a number of ways using animal models, and again, construct validity has been established using pharmacological, lesion and genetic models.

In a widely used assay of selective attention (see **Figure 2**), an animal is initially trained to respond when a target cue is present. The target cue can then be presented among an array of distracter stimuli, and depending on the number of shared features with the target, the animal will either use serial search (i.e., scan every item in the array in order to locate the target) or parallel search (the entire scene is processed in parallel, and the

target stimulus appears to "pop-out" of the array; Treisman and Gelade, 1980).

Selective attention can be inferred in zebrafish from their performance forming attentional sets in the reversal learning and ID-shift procedure described earlier (Parker et al., 2012a), but there is also promise to develop assays to examine more complex tasks examining serial and parallel processing in visual discrimination tasks. Zebrafish are effective at discriminating two stimuli in a variety of different conditions, under control of both aversive and appetitive contingencies (Levin and Chen, 2004; Colwill et al., 2005). In addition, further work that requires explicitly either topdown or bottom-up processing (Proulx and Serences, 2006) will provide an opportunity to explore the use of these attentional mechanisms in the zebrafish model.

Sustained attention can be assayed in humans using a continuous performance task (CPT; Rosvold et al., 1956). In animals, a variety of tests have been used (e.g., stop-signal task (Logan et al., 1997); go no-go task (Finn et al., 1999), but arguably the most useful has been the 5-choice serial reaction time task (5-CSRTT; Carli et al., 1983; Robbins, 2002), owing to the rich variety of parameters measurable in this assay. The 5-CSRTT (see **Figure 3**) tests the ability of animals to detect the presence of a briefly presented stimulus in one of five randomly ordered spatial locations following an inter-trial interval (ITI). Responses in the correct location during a limited time following the stimulus presentation (limited hold; LH) are conditionally reinforced with illumination of the magazine light, and subsequently food reinforcement, at the opposite end of the apparatus. Incorrect responses (errors of commission), anticipatory/premature responses during the ITI or failure to respond during the LH (errors of omission) are punished with a brief time-out. Subsequent trials are initiated with a nose-poke in the magazine. The 5-CSRTT can also be used to measure selective attention, as auditory or visual distracters can be added to the test environment during training or test sessions (Bari et al., 2008).

Sustained attention has been successfully demonstrated in zebrafish in two tasks (**Figure 4**). Initially, Bilotta et al. (2005) designed a task whereby fish were required to swim into one of three apertures. A light was illuminated in one of the chambers (see **Figure 4A**), and the barrier was lifted to allow the fish access to the choice area. The fish was reinforced for swimming into the

"fncir-07-00063" — 2013/4/5 — 12:30 — page 3 — #3

**FIGURE 3 | Measurement of sustained attention and impulse control in rodents:The 5-choice serial reaction time task.**

correct chamber. Incorrect choices were punished by confining the fish to the incorrect chamber for 30-sec with no food. Fish were found to perform well on this task, quickly reaching 80% correct choices with repeated testing.

Another version of the task increased the complexity by: (1) requiring the zebrafish to maintain attention of the three stimulus apertures prior to making a response, and (2) by requiring the fish to return to the opposite end of the tank to receive their reinforcer (**Figure 5B**; Parker et al., 2012b). Both tasks have the ability to examine aspects of sustained attention, such as increasing attentional load by varying the duration or intensity of the stimuli, and hold great promise in terms of assessing attentional processes in zebrafish. In addition, it will be possible to adapt either of these tasks to include visual or auditory distracters to attempt to produce a parametric assessment of selective attention in zebrafish.

## **IMPULSE CONTROL**

Impulsive behaviors can be broadly divided into two categories: those resulting from deficits in the ability to withhold responding (impulsive action) and those that result from deficits in decisionmaking (impulsive choice). Impulsive action can be operationally defined in terms of anticipatory responding on assays such as the 5-CSRTT (Robbins, 2002), the go/no-go task (Finn et al., 1999), or the stop-signal reaction time task (Logan et al., 1997); impulsive choice is exemplified by the choice of a small, immediate reward over a delayed, more substantial reward (delay-discounting; Winstanley et al., 2006; Dalley et al., 2008; Diergaarde et al., 2008). Both categories of impulsive behavior are reported in a number of psychiatric conditions such as ADHD (Barkley, 1997), substance abuse disorder (Belin et al., 2008), pathological gambling (Alessi and Petry, 2003), schizophrenia (Winstanley et al., 2003) and OCD (Brembs, 2009). Animal (e.g., rodent) models of impulse control

"fncir-07-00063" — 2013/4/5 — 12:30 — page 4 — #4

have been shown to have a good degree of construct validity, again using a combination of pharmacological, lesion and knockout studies.

Our lab has recently reported data pertaining to the performance of zebrafish in a three-choice version of the 5-CSRTT. Our version of the task is different from the rodent version, in that: 1) we use longer stimulus intervals (rodents = ∼0.5-sec, fish = 5– 10-sec), 2) we initiate the start of each trial by lifting the barrier to expose the stimulus apertures, while in rodents trials are initiated by nose-poking the magazine. Using this procedure, we demonstrated not only that zebrafish show similar rates of basal anticipatory responding on the task as rodents, but also that a low dose (0.025 mg/Kg i.p.) of amphetamine significantly reduced anticipatory responding during long ITI probe trials (low doses of psychostimulants reduce impulsivity in other animal models, and in humans; Robbins, 2002) whereas a saline injection had no effect (Parker et al., 2012b). In order to validate the procedure as a measure of impulse control, these findings need to be replicated with further pharmacological manipulations, and potentially with existing mutant lines. These preliminary findings are very encouraging, however, and we are in the process of automating the procedure tofacilitate validation studies andfuture screening programs. Measures of impulsive choice, as measured in rodents by delayed reinforcement procedures, are currently lacking in zebrafish.

## **NEURAL CIRCUITS MEDIATING BEHAVIORAL PHENOTYPES**

In this section we outline what is currently known about the neural circuits that underlie the behaviors described above and their conservation in zebrafish.

"fncir-07-00063" — 2013/4/5 — 12:30 — page 5 — #5

## **BEHAVIORAL FLEXIBILITY**

Evidence from lesion studies suggests that regions of the PFC (medial pre-frontal cortex [mPFC]; orbitofrontal cortex [OFC]; lateral PFC), striatum (specifically the nucleus accumbens [NAc] and the dorsal-medial striatum [DMS]) and thalamic nuclei are involved with various aspects of behavioral flexibility (Ragozzino et al., 1999; Brown and Bowman, 2002; Ragozzino, 2007). Collectively, these data have suggested that reversal learning in mammals is mediated by cortical- striatal-thalamo-cortical loops (**Figure 5**). There is also much evidence to suggest that reversal learning is modulated by both DA and serotonin (5-HT). For example, impairments of reversal learning can be induced by DA depletion in the NAc (6-OHDA lesions of NAc: Taghzouti et al., 1985), by inhibition of DA reuptake with amphetamine (Ridley et al., 1981) or by 5-HT depletion in the PFC (Clarke et al., 2004). **Figure 5A**summarizes the putative circuits of behavioral flexibility in mammalian systems.

The topography of the zebrafish brain differs from the mammalian brain, but homologues for the different brain regions have been identified. For example, both the mammalian and the zebrafish thalamic nuclei are located in the diencephalon (**Figure 6**), and homologues of midbrain regions such as the VTA (posterior tuberal nucleus; PTN) and NAc (ventral [Vv] and dorsal [Vd] telencephalic nuclei; Rink and Wullimann, 2002; Panula et al., 2010) have been determined. Recently, Mueller et al. (2011) have also identified the central region of the dorsal pallium (area Dc in **Figure 6**) as a potential homologue of the isocortex, that in mammals encompasses the above-mentioned cortical regions. There are a number of neurochemical pathways relevant to behavioral flexibility that have a good degree of homology. For example, the ascending midbrain DA pathways have been well characterized using tyrosine hydroxylase (TH) immunohistochemistry (Rink and Wullimann, 2001, 2002; Filippi et al., 2010), with a number of putative functional homologues being identified owing to their neuronal connections and projections (**Figure 6**). Much of the evidence pertaining to the cholinergic, DAergic and 5-HTergic neural clusters in the zebrafish brain has been generated from extensive immuno-staining of relevant cell bodies. There is little evidence of how the systems functionally interact, and no direct evidencefor a homologous circuitfor the cortico-striatal-thalamocortical loops. Specifically, whether or not the neural clusters are reciprocally connected remains to be seen.

### **SELECTIVE ATTENTION**

**Figure 5B** summarizes the putative neural circuits of selective attention in mammalian systems. The cholinergic system plays a central role in selective attention (Robbins, 1997). Cholinergic neurons project widely to such basal forebrain structures as the habenula (Claudio Cuello et al., 1978) and the striatum (Woolf and Butcher, 1986), releasing ACh at various synaptic terminals across all layers of the cortex via activation of cholinergic receptor sub-types (Sarter and Bruno, 1997). ACh binds to two distinct categories of cholinergic receptors: G-protein coupled metabotropic muscarinic receptors (mAChR) and ligand-gated ionotrophic nicotinic receptors (nAChR), both of which are implicated in attention (Noudoost and Moore, 2011). In addition, the pedunculopontine tegmental nucleus (PPTN) may play a dual role in selective attention pertaining to the processing of saliency and reward-cues. Specifically, cholinergic outputs from the PPTN synapsing on lateral geniculate nucleus (LGN) cells expressing nAChRs and mAChRs regulate saliency, and those synapsing on substantia nigra pars compacta (SNc) DAergic cells regulate reward processing (Kobayashi and Isa, 2002).

The role of the DA system in attention is also well established (Nieoullon, 2002). For example, people with Parkinson's disease (characterized by a loss of nigrostriatal DA neurons; Owen et al., 1993) and ADHD (characterized by reductions in PFC DA; Barkley, 1997) both show deficits in selective attention, as do people with schizophrenia (characterized by increases in D2-like receptors in the striatum; Seeman et al., 1993). Although not universally accepted, there is some evidence that ACh modulation may relate more to aspects of saliency (Asadollahi et al., 2010; Knudsen, 2011), and DA more to motivated search. As such, it may be that the roles of ACh and DA are, respectively, related to bottomup and top-down processing in a dissociated, or at least partially dissociated manner (Noudoost and Moore, 2011).

In terms of homologous brain regions involved in cholinergic signalling, the cortical regions thought to be involved with selective attention are relatively well conserved in zebrafish, although the topography is, again, somewhat different (**Figure 6**). Thus, the cholinergic projections from the PPTN to the brainstem, habenula and thalamic (e.g., LGN) regions in rodents are mirrored by projections from the superior reticular nucleus (SRN) to the brainstem, habenula and subpallium in fish. As discussed above, the neurotransmitter systems thought to modulate selective attention (DA, ACh) are well conserved in zebrafish (see **Figure 6**). For example, the mAChRs and nAChRs have been identified in zebrafish and their binding characteristics and expression patterns described (Zirger et al., 2003). Similarly, the DA system has been characterized and the homologues of key components of the reward pathway identified as discussed above (Filippi et al., 2010). In the light of this, neuromodulatory influences may be conserved between the species.

### **SUSTAINED ATTENTION**

Thefrontal cortices (frontal executive system) are thought to mediate sustained attention (Barkley, 1997). Indeed, some theories of ADHD (which is characterized, in part, by difficulties in sustained attention) suggest that the symptoms may be caused by delayed cortical maturation (Loo and Barkley, 2005). Although the circuits underlying sustained attention are less clearly defined, lesions studies have indicated brain regions involved (see **Figure 5C**). Notably, the PPTN has outputs via the SNc to striatum and to PFC suggesting a role of the nigrostriatal DA pathway in some aspects of sustained attention, perhaps relating to motor control during tasks requiring sustained attention. In addition to the frontal cortices, the striatum appears to be crucially linked to aspects of sustained attention (Barkley, 1997).

The cholinergic system, including cholinergic neuronal projections and cholinergic receptors (mAChR and nAChR), plays a crucial role in sustained attention. Cortical ACh is released during tasks requiring sustained attention (Arnold et al., 2002), and as the task increases in difficulty, the release of ACh increases (Himmelheber et al., 2000). The DAergic system has also been

"fncir-07-00063" — 2013/4/5 — 12:30 — page 6 — #6

"fncir-07-00063" — 2013/4/5 — 12:30 — page 7 — #7

#### **FIGURE 6 | Continued**

**Schematic sagittal view comparing dopaminergic (green), serotonergic (red), and cholinergic (blue) neuronal populations in zebrafish (upper) and rat (lower) brains.** Nomenclature for serotonergic and dopaminergic populations were based on their rostrocaudal distribution in the adult rat and zebrafish brain previously described (Schweitzer and Driever, 2009; Panula et al., 2010) allowing comparison between cell body distribution in adult brains. **(A)** Cell body distribution (adapted from Manger et al., 2002; Butcher and Woolf, 2003; Mueller et al., 2004; Schweitzer and Driever, 2009; Panula et al., 2010). Corresponding **(A,B)** nomenclature for dopaminergic and Serotonergic cell bodies derived from Panula et al. (2010) and Schweitzer and Driever (2009). Dopaminergic populations (zebrafish): A8: parvocellular preoptic nucleus, posterior part (PPp); A9: periventricular nucleus of posterior tuberculum (TPp); A10: periventricular hypothalamus and posterior tuberculum (PTN); A11: posterior tuberculum (PTN); A13: anterior, intermediate, ventrolateral, and ventromedial thalami nuclei (A, I, VM, and VL); A14: parvocellular preoptic nucleus, anterior part (PPa); A15: parvocellular preoptic nucleus, anterior part (PPa); A16p: ventral telencephalic nuclei (Vv, Vd and Vs); A16a: olfactory bulb (Ob). Dopaminergic populations (rat): A8: midbrain reticular formation (MRF); A9: ventrolateral midbrain (VLM); A10: ventral midbrain tegmentum (VTAe); A11: posterior periventricular nucleus and intermediate periventricular nucleus (PVNP and PVNI); A12: hypothalamic arcuate nucleus (ARH); A13: mammillothalamic tract (MTT); A14: anteroventral periventricular nucleus (AVPV); A15: Anteroventral periventricular nucleus (AVPV); A16: olfactory bulb (OB). Serotonergic populations (zebrafish): B1-B2: caudal raphe complex; B3-B9: rostral raphe complex; BPVOa: paraventricularorgan, anterior part (PVOa); BPVOi: paraventricular organ, intermediate part (PVOi); BPVOp paraventricularorgan posterior part (PVOp). Serotonergic populations (Rodent): B1: raphe pallidus (RPa); B2: raphe obscurus (NRO); B3: raphe magnus (NRM); B4: vestibular nucleus (VN); B5: pontine raphe nucleus (PRN); B6: medial longitudinal fasciculus (MLF); B7: dorsal raphe nucleus (DRN); B8: median raphe nucleus (MRN); B9: median raphe nucleus (MRN).  Cholinergic populations (rat): ldt: lateral dorsal tegmental nucleus; si: substantia innominate; hdb: horizontal diagonal band nucleus; vdb: vertical diagonal band nucleus; bas: nucleus basalis; ms: medial septal nucleus (MS); md: medial habenula (MH); III: oculomotor nucleus (IIIn); IV: trochlear nucleus (IVn); VII: facial nucleus (VIIn); X: dorsal motor vagus nucleus (Xn); XII: hypoglossal nucleus (Xn). SP: Subpalium (Sp). **(B)** Schematic drawing illustrating the location of dopaminergic projections in adult zebrafish and rat brains (sagittal view; adapted from Schweitzer and Driever, 2009). **(C)** Schematic drawing illustrating the location of serotoninergic projections in adult zebrafish (adapted from Gaspar and Lillesaar, 2012) and rat brains (adapted from Di Giovanni et al., 2008; sagittal view). **(D)** Schematic drawing illustrating the location of cholinergic neuron projections from PPT in adult rats (adapted from (Manger et al., 2002)) and predicted projections from zebrafish SRN to subpallium and habenula. D, dorsal telencephalic area; Dc, caudal dorsal telencephalic area; CTX, cerebral cortex CB, cerebellum; Ha, habenula; Hc, caudal zone of periventricular hypothalamus; HIP, hippocampus; Hv, ventral zone of periventricular hypothalamus; IMRF, intermediate reticular formation; IPN, interpeduncular nucleus; IRF, inferior reticular formation; LC, locus coeruleus; LH, lateral hypothalamic nucleus; LR, lateral recesses of the diencephalic ventricle; LVII, facial lobe; LX, vagal lobe; NIII, oculomotor nucleus; OB, olfactory bulb; ON, optic nerve; OBN, olfactory bulboptic nerve; PTN, posterior tuberculum; PVO, paraventricular organ, anterior part; PVOi, paraventricular organ, intermediate part; BPVOp, paraventricular organ posterior part; SRF, superior reticular formation; Vd, dorsal telencephalic area; Vv, ventral telencephalic area; TeO, optic tectum; TH, thamalus.

strongly linked to sustained attention, in particular relating to the dissociable roles of D1 and D2 receptors in the mPFC. **Figure 5C** summarizes the putative neural circuits of sustained attention.

As mentioned above, homologous regions exist and the midbrain DAergic system has been well characterized in zebrafish. In addition to this, the cholinergic system in zebrafish has been characterized with choline acetyltransferase (ChAT) immunohistochemistry (Mueller et al.,2004; **Figure 6**). Cholinergic cell bodies are found in the caudal raphe complex of both mammals and zebrafish (**Figure 6**). Of particular note here, zebrafish have an ascending SRN cholinergic system which is a putative homologue for the mammalian PPTN, lesions of which are known to impair sustained attention in rats (Kozak et al., 2005).

## **IMPULSE CONTROL**

Impulse control and motivational circuits are inextricably linked. Motivational circuits have been extensively characterized (Dalley et al., 2008; **Figure 5D**). As is the case with behavioral flexibility, lesions to the medial dorsal, but not anterior, thalamic nucleus, cause increases in premature responding (Chudasama and Muir, 2001). The data strongly suggest that impulsivity, at least that form of impulsivity measured by the 5-CSRTT, is mediated by DAergic and 5-HTergic cortico-striatal-thalamo-cortical loops (Robbins, 2002). Clearly this suggests a role for catecholaminergic systems in impulsivity, and indeed high doses of systemic amphetamine administration increases premature responding, an effect that is reversed by 6-OHDA lesions of the NAc (Cole and Robbins, 1989). In addition, systemic atomoxetine, a noradrenergic reuptake inhibitor, reduces impulsivity (Robinson et al., 2008). Finally, rats characterized as "high-impulsive" on the 5-CSRTT have reduced DA D2/3 receptors in the NAc (Dalley et al., 2007). **Figure 5D** summarizes the putative circuits of impulse control in mammalian systems.

The neural circuits currently hypothesized to modulate impulse control (e.g., ascending midbrain DA pathways and raphe complex 5-HT pathways; **Figure 6B**) are present in zebrafish, or at least, putative functional homologues exist (**Figure 6**). For example, the caudal raphe complex is well conserved between species, as are the 5-HT projections from this region to prefrontal regions in rats and dorsal pallial regions in the zebrafish. In addition, homologues for the VTA (PTN), NAc (Vv and Vd; Rink and Wullimann, 2002; Panula et al., 2010) and area Dc (Mueller et al., 2011) have been identified and DA projections in these regions are similar. In the zebrafish brain DAergic and 5-HTergic projections from the pallium to the thalamic nuclei, in addition to DAergic projections from the telencephalic nuclei to the pallium (see **Figure 6**), suggest similar patterns of connectivity to mammalian brains.

## **FUTURE PROSPECTS: HOW CAN ZEBRAFISH CONTRIBUTE TO THE UNDERSTANDING OF EXECUTIVE FUNCTION AND THE ETIOLOGY OF PSYCHIATRIC DISEASE?**

To date researchers interested in using zebrafish to study executive function have focused on the development of appropriate behavioral paradigms. Now that many assays have been established zebrafish are well placed to address key questions relating to the control of these behaviors, particularly in the areas of impulse control and attention, and the etiology of disease: (1) Which neural circuits are involved and how do they develop? (2) What are the genetic factors that influence impulse control and attention?

"fncir-07-00063" — 2013/4/5 — 12:30 — page 8 — #8

(3) What are the cell biological processes by which genetic factors act to influence these behaviors and contribute to behavioral disease? The primary advantages zebrafish have in the search for knowledge in these areas are their transparency, which facilitates *in vivo* analysis of the development and functioning of neural circuits controlling behavior, and the ability to perform large scale pharmacological and genetic screens.

#### **DISSECTION OF THE NEURAL CONTROL OF BEHAVIOR**

The information regarding brain regions and pathways involved in the rodent behaviors described above were in large part obtained using lesion studies and pharmacological manipulations. These approaches are relatively crude as manipulations often affect surrounding cell types and processes or have slow reversibility (Mei and Zhang, 2012). In contrast, the use of optogenetic techniques that combine the use of light-controlled reporters and manipulators of neuronal activity with genetic targeting, allows more precise dissection of the neural control of behavior. A number of different optogenetic constructs for manipulating neuronal activity are available for use in fish as in other species. For example, channelrhodopsin cation channels can activate neurons by depolarising the membrane potential upon activation by light, whereas halorhodopsin and bateriorhodopsin channels act as light sensitive chloride and proton pumps capable of hyperpolarizing the membrane thus inhibiting action potentials. Although utilized in many model organisms, zebrafish are particularly well suited to the application of optogenetic techniques. In transparent larval and, to lesser extent, adult *casper* (White et al., 2008) forms, we have the ability to drive expression in specific cell types using GAL4:UAS constructs (Davison et al., 2007; Scott et al., 2007; Asakawa and Kawakami, 2008; Asakawa et al., 2008) or transposon technologies (Petzold et al., 2009). This provides an almost entirely non-invasive method for visualizing and modulating neuronal activity at even the single cell level and examining the effect on behavior. By comparison, in other animal systems, such as murine or primate models, holes must be drilled into the skull and fiber optic cables inserted to have access to the brain and control behavior *in vivo* with light optics (Cao et al., 2010). As the application of optogenetic techniques to address development and functioning of neural circuits in zebrafish have been extensively discussed elsewhere (Schoonheim et al., 2010; Del Bene and Wyart, 2012; Portugues et al., 2012; Umeda et al., 2012), in the section below we summarize more recent advances in technologies to visualize developing neuronal circuits.

Recent technological advances based on the use of genetically encoded GFP variants allow individual projections (*Brainbow*; Pan et al., 2011) and synaptic contacts GFP reconstituted across synaptic partners (GRASP) to be resolved, and activity within neuronal circuits (GCaMP) to be followed using fluorescence microscopy. *Brainbow* relies on combinatorial expression of several fluorophores (XFPs) to label individual neurons and their projections. Using Cre-lox recombination technology under the control of neuron specific promoters, each individual neuron expresses a random combination of each of up to four different XFPs to generate a specific fluorescence signal. As each neuron expresses the four XFPs at different levels, up to 100 different spectra can be obtained. Using this technology it is possible to trace the neuronal projections and formation of neuronal circuits in developing embryos and larvae. Pan et al. (2011) used this approach to map the zebrafish trigeminal projections.

"GFP reconstituted across synaptic partners" technology developed in *c. elegans* (Feinberg et al., 2008) can be used to trace the formation of synaptic contacts at high resolution *in vivo*. GRASP involves the expression of complementary fragments of GFP tethered to extracellular domains of transmembrane carrier proteins on pre- and post-synaptic membranes. The individual fragments of GFP are not fluorescent, but the formation of a synapse brings the two fragments into close proximity allowing reconstitution of the fluorescent molecule. Although application of this technique to zebrafish circuits has yet to be published, it has been used to map neuronal connections in *c. elegans*, *Drosophila* and mice (Feinberg et al., 2008; Arenkiel and Ehlers, 2009; Kim et al., 2011) and has the potential to trace synapse formation *in vivo* in wild-type and behaviorally mutant zebrafish.

In addition to tracing neuronal projections and synapse formation, fluorescence, including GFP technologies, have been used to monitor activity within neuronal populations in living behaving larval zebrafish. For example, the group of Rainer Friedrich (Li et al., 2005) has used whole brain calcium imaging to track activity within neuronal olfactory circuits as larval fish respond to changes in olfactory cues. Herwig Baier's group (Del Bene et al., 2010) used GCaMP reporter constructs under the control of neuron specific promoters to identify neural circuits involved in processing visual information. Using a similar approach, Koichi Kawakami's group (Muto and Kawakami, 2011; Muto et al., 2011, 2013) have identified circuits involved in spontaneous motor behavior and perception in embryonic zebrafish. When coupled with optogenetic approaches to manipulate activity in specific cells, these techniques provide a powerful means of dissecting neuronal circuits controlling behavior. Another particularly elegant larval assay that could be used to address the neurobiology of selective attention was recently described. Bianco et al. (2011) tracked the eye convergence and body position in 7dpf larvae, partially restrained in agarose, in response to different sized virtual stimuli. They found that the larvae tracked small moving spots, adopted a J-bend of their tail (the body shape that precedes prey capture; McElligott and O'Malley, 2005) and showed eye convergence on the target. This initial evidence of oculomotor processing of prey provides some evidence of a basic form of saliency-based selective attention, which may hold great promise for the development of tests of executive function in zebrafish in the future. As discussed by Bianco et al. (2011), by combining the assay with functional imaging of genetically encoded calcium indicators (Higashijima et al., 2003), techniques to manipulate circuits (Douglass et al., 2008; Janovjak et al., 2010; Schoonheim et al., 2010), and targeted laser-ablations (McLean et al., 2007; Satou et al., 2009; Burgess et al., 2010) it will be possible to identify the neuronal circuits controlling this, and similar, behavior.

Although the majority of these fluorescence techniques have to date only been applied to larval fish, advances in multiphoton confocal technology and computer processing raises the possibility of performing similar studies in juvenile and adult fish in the

"fncir-07-00063" — 2013/4/5 — 12:30 — page 9 — #9

future. These imaging approaches coupled with the large-scale mutagenesis analysis possible in zebrafish have unprecedented potential to extend understanding of the cellular and molecular bases of behavior

### **FORWARD GENETIC SCREENING**

The ability to perform forward genetic screens for behavioral phenotypes also has great potential to advance understanding of the neurobiology of behavior. Forward genetic mutagenesis screens in zebrafish have been widely used to identify mutant alleles affecting developmental phenotypes. The classic three-generation mutagenesis screen for recessive alleles looks for families in which 25% of the F3 offspring show a given phenotype. This approach works well for recessive (or dominant) alleles of major effect but has, thus far, proved less effective for complex behavioral phenotypes likely to be governed by multi-allelic variations, each of minor effect and variable penetrance. Nonetheless, forward genetic screens for behavioral phenotypes have been undertaken. Darland and Dowling (2001) and Ninkovic et al. (2006) performed screens for cocaine and amphetamine-induced place preference, respectively. Both isolated lines of fish with differential drug seeking behavior, but neither have successfully isolated the causal mutations, possibly due to difficulties in unambiguously identifying the mutant carrier; the performance of control individuals often falls within the range of affected individuals and vice versa (Jain et al., 2011) making linkage analysis difficult.

Population based breeding and selection, or GFP insertion techniques can be used to address this problem. For example, Jain et al. (2011) used a "phenotyping by segregation" approach, based on commonly used breeding and selection strategies, to map the hypersensitive zebrafish *houdini* mutant. This strategy is attractive as it allows for fine mapping of subtle phenotypes that may have variable penetrance in the general population. An alternative approach taken by Petzold et al. (2009) used fluorescently tagged gene breaking transposons to mutagenize zebrafish. These transposons permit visual sorting of carriers from non-carriers (fluorescent vs. non-fluorescent larvae) and have the advantage of allowing rapid cloning of the mutagenized gene. Petzold et al. (2009) successfully used this insertional mutagenesis approach to identify two genes involved in the behavioral response of larval fish to nicotine.

Application of such breeding and selection-based and insertional mutagenesis screening approaches to the adult behavioral assays outlined above may lead to the identification of novel genes contributing to complex behavioral phenotypes. Such studies will make a valuable contribution to complement genome wide association studies (Sullivan,2010) and analyses of copy number variants

#### **REFERENCES**

(Cook and Scherer, 2008) aimed at understanding the genetics of psychiatric disease.

## **IDENTIFYING CELL BIOLOGICAL PROCESSES AFFECTING COMPLEX BEHAVIORS**

Genome-wide association studies in humans are excellent tools for identifying genetic variations associated with psychiatric disorder phenotypes (Mitchell and Porteous, 2009; Ersland et al., 2012). In some instances these studies are even being able to identify endophenotypes associated with multiple psychiatric disorders (Hall and Smoller, 2010; Consortium, 2013). However, while being able to identify genetic markers associated with a particular disorder, they are not able to establish which variations are of causal effect. The ability in zebrafish to generate targeted knockouts of candidate genes using TALEN technology (Sander et al., 2011) offers a cost effective and convenient means of investigating which of the candidate alleles identified in human GWAS studies are causally linked to behaviors. Further, once an allele of effect is identified, zebrafish provide an ideal model system in which to investigate the neurodevelopmental and cellular processes affected.

## **CONCLUSION**

In this review, we have discussed the potential for using zebrafish to uncover some of the molecular and cellular processes related to psychiatric disorder, in particular relating to disorders of executive function. These are exciting times for zebrafish researchers. New assays of subtle behavioral phenotypes are fast being developed and replicated in different laboratories, and validation of these phenotypes is underway and progressing well. Given the huge repertoire of genetic tools available and the ever-expanding mutant resource, zebrafish will soon become one of the leading animal models in behavioral neuroscience. Even in the event that there are significant differences in anatomy or connectivity, many of the behaviors we have discussed are extensions of evolutionarily ancient reward and impulse control processes that appear to have conserved neurochemical pathways. In this regard, understanding the molecular mechanisms regulating these processes in fish will still give insight into regulation in mammals. As a final thought, there still remains much debate over how best to describe even simple neural circuits, and at this point no vertebrate system, regardless of technological advances, can come close to dealing with this issue (Yuste, 2008). Even in the age of the Human Connectome Project, the complexities of characterizing functional neural circuits should not be underestimated (Koch, 2012).

rats performing a sustained attention task versus behavioral control tasks that do not explicitly tax attention. *Neuroscience* 114, 451–460.

Gal4-UAS system in zebrafish. *Dev. Growth Differ.* 50, 391–399.

"fncir-07-00063" — 2013/4/5 — 12:30 — page 10 — #10

action: contingency and incentive learning and their cortical substrates. *Neuropharmacology* 37, 407–419.

to social defeat and antidepressant action. *J. Neurosci.* 30, 16453– 16458.

trait impulsivity and cocaine reinforcement. *Science* 315, 1267–1270.

Shen, K., et al. (2008). GFP reconstitution across synaptic partners (GRASP) defines cell contacts and synapses in living nervous systems. *Neuron* 57, 353–363.

"fncir-07-00063" — 2013/4/5 — 12:30 — page 11 — #11

optical inhibition of neuronal firing. *Nat. Neurosci.* 13, 1027–1032.

*Proc. Natl. Acad. Sci. U.S.A.* 108, 5425–5430.

"fncir-07-00063" — 2013/4/5 — 12:30 — page 12 — #12

engineered TALENs. *Nat. Biotechnol.* 29, 697.

as a tool for in vivo transplantation analysis. *Cell Stem Cell* 2, 183–189.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 08 January 2013; accepted: 19 March 2013; published online: 08 April 2013.*

*Citation: Parker MO, Brock AJ, Walton RT and Brennan CH (2013) The role of zebrafish (Danio rerio) in dissecting the genetics and neural circuits of executive function. Front. Neural Circuits 7:63. doi: 10.3389/fncir.2013.00063*

*Copyright © 2013 Parker, Brock, Walton and Brennan. This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

"fncir-07-00063" — 2013/4/5 — 12:30 — page 13 — #13

## The first mecp2-null zebrafish model shows altered motor behaviors

## *Thomas Pietri 1,2,3\*, Angel-Carlos Roman4†, Nicolas Guyon1,2,3†, Sebastián A. Romano1,2,3, Philip Washbourne5, Cecilia B. Moens 6, Gonzalo G. de Polavieja4 and Germán Sumbre1,2,3*

*<sup>1</sup> Ecole Normale Supérieure, Institut de Biologie de l'ENS, Paris, France*

*<sup>2</sup> Inserm, U1024, Paris, France*

*<sup>3</sup> CNRS, UMR 8197, Paris, France*

*<sup>4</sup> Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid, Spain*

*<sup>5</sup> Institute of Neuroscience, University of Oregon, Eugene, OR, USA*

*<sup>6</sup> Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA*

#### *Edited by:*

*Rainer W. Friedrich, Friedrich Miescher Institute for Biomedical Research, Switzerland*

#### *Reviewed by:*

*Emre Yaksi, VIB, Belgium Caroline H. Brennan, Queen Mary University of London, UK*

#### *\*Correspondence:*

*Thomas Pietri, Ecole Normale Supérieure, Institut de Biologie de l'ENS, 46 rue d'Ulm, 75005 Paris, France e-mail<EMAIL_ADDRESS>
*†These authors have contributed equally to this work.*

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder and one of the most common causes of mental retardation in affected girls. Other symptoms include a rapid regression of motor and cognitive skills after an apparently early normal development. Sporadic mutations in the transcription factor MECP2 has been shown to be present in more than 90% of the patients and several models of MeCP2-deficient mice have been created to understand the role of this gene. These models have pointed toward alterations in the maintenance of the central nervous system rather than its development, in line with the late onset of the disease in humans. However, the exact functions of MeCP2 remain difficult to delineate and the animal models have yielded contradictory results. Here, we present the first *mecp2*-null allele mutation zebrafish model. Surprisingly and in contrast to MeCP2-null mouse models, *mecp2*-null zebrafish are viable and fertile. They present nonetheless clear behavioral alterations during their early development, including spontaneous and sensory-evoked motor anomalies, as well as defective thigmotaxis.

**Keywords: zebrafish, motor behavior, Rett syndrome, mecp2, early development, thigmotaxis**

## **INTRODUCTION**

Epigenetic regulations of gene expression play a crucial role in brain development and maturation. Accordingly, alterations of factors involved in epigenetic regulation at the level of gene transcription lead to critical defects in brain function. Methyl-CpG-binding protein 2 (MECP2), a basic transcription factor, is a major epigenetic regulator. It was first described as a global repressor of transcription (Lewis et al., 1992). More recently, apart from its well documented repressor activity, MECP2 seems to act as a gene expression activator (Yasui et al., 2007; Chahrour et al., 2008), to modulate gene expression by long range chromatin remodeling (Skene et al., 2010) and to regulate RNA splicing (Young et al., 2005).

MeCP2, present in all vertebrate species, appears to be an innovation of this phylum. It shows a high level of conservation among mammals with, for instance, more than 95% protein sequence identity between human and mouse, and to a lesser extent, between humans and zebrafish (48%). In mammals, like in zebrafish, MeCP2 appears to be expressed throughout the body in multiple organs, and particularly enriched in the nervous system. Its expression in the nervous system is first detected during early embryonic development and it is highly expressed in postmitotic neurons (Shahbazian et al., 2002a; Curado et al., 2007). The structure and expression patterns of MeCP2 in zebrafish and mammals are similar (Coverdale et al., 2004), suggesting strong evolutionary pressure and therefore probable conserved functions.

Alterations of MECP2 functions have been shown to be causative of several neurodevelopmental diseases associated with autism spectrum disorders. In particular, sporadic mutations of MECP2 have been uncovered in more than 90% of patients diagnosed with Rett syndrome (RTT, Amir et al., 1999). At the clinical level, patients with RTT are asymptomatic during the first 6 to 18 months of life, appearing to develop normally. Subsequently, a period of stagnation precedes a net regression of the acquired skills. It is during this period that mental retardation appears and psychomotor skills degrade. Some patients also display seizures, abnormal cardiac, or breathing cycles (Matsuishi et al., 2011).

Various mouse models of RTT have been created, ranging from null-MeCP2 mutations to specific point ones mimicking those in humans.  These different models point toward alterations of synaptic and neural circuits maturation and maintenance, in particular an imbalance between excitation and inhibition (Dani et al., 2005). Interestingly, this imbalance could affect various brain regions and at different stages of development (Blue et al., 2011; Kron et al., 2012). In addition to the effect on neurons, it has been recently shown that MeCP2 dysfunction may also perturb glial cells (Ballas et al., 2009; Lioy et al., 2011). Overall, these findings suggest that MeCP2 functions appear to be cell type and developmental stage dependent, thus impeding the delineation of the entire spectrum of MeCP2 functions and the association of a particular functional alteration to a specific phenotype.

Zebrafish has recently gained much attention as a vertebrate model for human neurodevelopmental and neurodegenerative diseases (Panula et al., 2006; Beattie et al., 2007; Morris, 2009; Brennan, 2011; Kabashi et al., 2011; Steenbergen et al., 2011; Xi et al., 2011). Importantly, zebrafish produce a large number of progeny, with fast and external embryonic and larval development. They have a large repertoire of well-studied motor behaviors (for recent review, see Saint-Amant, 2010). Molecular manipulations of its genome can be achieved through forward genetic screening of mutagenized fish (Draper et al., 2004) or genome editing methods (Huang et al., 2011). Overall, these features have established zebrafish as a powerful and complementary vertebrate model for behavioral and genetic analysis, and more recently as an invaluable human disease model.

Here, we introduce the first zebrafish model of RTT, in which mecp2 function was abolished. A kinematic analysis of motor activity in embryos and young larvae reveals alterations of spontaneous, sensory-evoked and thigmotaxic behaviors.

## **MATERIALS AND METHODS**

#### **ANIMALS**

AB adult, larva, and embryo zebrafish, from the University of Oregon Zebrafish Facility, were maintained at 28.5◦C on a 14–10 h on/off light cycles at the Institut de Biologie de l'Ecole Normale Supérieure zebrafish facility. Embryos and larvae were grown accordingly to Westerfield (2000). All experimental procedures were performed at room temperature (21–23◦C). Zebrafish *mecp2Q63*<sup>∗</sup> mutation was generated through *N*-ethyl-*N*-nitrosourea (ENU)-mutagenesis and selected by TILLING as previously described (Draper et al., 2004). As ENU-mutagenesis generates random mutations throughout the genome, heterozygote *mecp2* mutant fish were outcrossed several times in the AB background to remove off-target mutations. Mutant fish were identified by PCR using DNA extracted from fin clip (the primers 5 -AAAGGAAAGGCATGATGTGG-3 and 5 -GTATCGCCAACCTTTTGGAA-3 flank the position of the mutation), followed by sequencing. To keep the closest genetic background between different genotypes in the experiments, heterozygote *mecp2* mutant embryos and larvae were generated by outcrossing homozygote *mecp2* mutants with AB wild-type fish. Wild-type, heterozygote and homozygote *mecp2* mutant fish are, respectively, labeled WT, Het, and Mut in the figures. Morphological assessment of aged-matched wild-type, homozygote and heterozygote mecp2 mutant embryos and larvae did not reveal any difference, indicating that the rate of development is similar in the three groups of embryos and larvae. Accordingly, we used hours post fertilization (hpf) and days post fertilization (dpf) to stage embryos and larvae, respectively.

All procedures were carried out in compliance with the guidelines of the University of Oregon and Le Comite d'Ethique pour l'Experimentation Animale Charles Darwin.

### **SYNTENY MAPPING**

Synteny mapping was performed by comparing the organization of the human genomic region bearing the MECP2 gene, with the zebrafish genome. To carry out this analysis, BLASTp searches were implemented using human proteins, inferred from genes present in this region, against the zebrafish database. Each first hit in the searches where then confirmed by a reciprocal BLASTp search. Only the reciprocal best hits of BLASTp searches were considered.

## **WESTERN BLOTTING**

Adult brain was homogenized in 1× RIPA extraction buffer (Cell Signaling) complemented with protease inhibitor cocktail tablets (Roche) and incubated on ice for 45 min, then centrifuged at 14,000 g for 5 min. The supernatant was collected and the protein concentration was measured with the BCA Protein Assay Kit (Pierce). Samples were then boiled in Laemmli buffer, separated on SDS-PAGE gel, transferred to nitrocellulose membrane and successively probed with a chicken anti-human MeCP2 C-terminal antibody (gift from Dr. LaSalle) at a dilution of 1µg/ml and visualized with a goat anti-chicken IgY HRP-coupled antibody (Jackson Laboratories) at a dilution of 0.1 µg/ml.

## **KINEMATIC ANALYSIS OF MOTOR BEHAVIORS**

Experiments were performed on 25 hpf, 51 hpf embryos, and 6 dpf larvae. The 25 hpf embryos were dechorionated at least one hour before the experiments. All embryos and larvae were acclimatized to room temperature for at least 20 min before the beginning of the experiments. To estimate the rate of spontaneous contractions, the freely moving 25 hpf embryos were recorded under a stereo-microscope (Leica LZMFIII) using a monochrome digital camera (PixeLINK PL-A741) at 20 Hz. over a period of 5 min. Fifty one hpf larvae were subjected to light mechanical stimulation using an eyelash either on the side of the trunk or on the head of the larvae. Stimulations were applied 5–7 times separated by at least 1 s. Larva motor responses were recorded at 100 Hz. The startle responses induced by the stimulations were analyzed with ImageJ software. Only the duration of complete coilings were measured. These were defined as the period between the first deviation of the trunk from its resting state until its return to its initial position. In case of multiple contractions after a stimulation, only the first coiling period was taken into account. To monitor the spontaneous swimming activity, 6 dpf wild-type and mecp2-mutant larvae were randomly placed in a custom-made Plexiglas 35-well plate (15 mm diameter × 0.5 mm height) filled-in with 500µl embryo medium at room temperature and let to habituate for 20 min before the experiment. Homogeneous illumination was provided by an electroluminescent panel (MiniNeon, France). Spontaneous motor behavior was monitored with a ImagingSource DMK 21BF04 camera at 30 Hz for 15 min. Custom Matlab scripts (MathWorks) were developed to compute the trajectories, activity, velocities, and the thigmotactic behavior. All Matlab scripts are available upon request. Larva activity was defined as the percentage of time in which movement was detected. Activity bouts and resting times were calculated as the periods of movement and between consecutive movements, respectively. Both were fitted to a power law of the *<sup>Y</sup>* <sup>=</sup> *aX*−*<sup>k</sup>* form. Thigmotactic behavior analysis described the relative position of the larvae within the well over time. For this purpose, we divided the wells in two zones: an

inner zone at the center of the well covering 36% of the total area, and an outer ring region covering the remaining 64% (see **Figure 3B**). To take into account potential alterations of motor functions in mecp2-null larvae, we analyzed the kinematics of a subset of swimming periods. Criteria for the selection of these subsets of trajectories are detailed in the results section.

### **STATISTICS**

Results are presented by classical box-and-whisker diagrams with the median as the central red mark, the first quartile (q1) and third quartile (q3) for the edge of the box, and the extreme data points represented by the whiskers in the range of q1–1.5∗(q3–q1) to q3 + 1.5∗(q3–q1). Statistical significance was assessed using the Kolmogorov–Smirnov test.

## **RESULTS**

## **ZEBRAFISH mecp2 IS AN ORTHOLOG OF HUMAN MECP2**

Tetrapod vertebrates possess one copy of the *mecp2* gene located on the X chromosome. However, during evolution, teleost fish underwent an additional round of full genome duplication, leading to the possibility that in zebrafish two copies of the *mecp2* gene are present. Synteny analysis was therefore carried out to compare the genomic region of the human chromosome X flanking the MECP2 gene to the zebrafish genome. After reciprocal BLASTp searches with the sequences of the human proteins inferred from the genes flanking human MECP2, we found two syntenic regions in chromosome 8 and chromosome 11 (**Figure 1A**), in which zebrafish genes orthologous to the human genes flanking MECP2 were detected. Moreover, several paralog genes were present in these two zebrafish genomic regions, covering 2.07 megabases (Mb) in chromosome 8 and 0.07 Mb in chromosome 11 (only the cxxc1 and hcfc1 paralog group has been indicated for clarity) and flank the *mecp2* gene in chromosome 8. The comparison of the genomic organization of these two regions enabled us to conclude that the zebrafish genome has retained only one copy of the *mecp2* gene which is the ortholog of the human MECP2.

## **CHARACTERIZATION OF THE** *mecp2Q63***<sup>∗</sup> MUTATION**

Screening of the two last exons of *mecp2* in ENU-mutagenized zebrafish sperm allowed us to recover by TILLING a C to T transition near the 5 part of the *mecp2* coding sequence, at position 187, causing a non-sense mutation and putatively truncating the protein at position 63. The mutant allele would then encode a form of the protein lacking both the crucial methyl-CpG binding (MBD) and the transcription repressor (TRD) functional domains (**Figure 1B**) most probably engendering a full loss of function of mecp2. C to T transition in homozygote mutants was confirmed by sequencing *mecp2* PCRamplified genomic DNA fragments, showing the creation of a non-sense amber codon in place of a glutamine codon in position 187/189 (**Figure 1C**). F1 generation was outcrossed in the AB background to remove potential mutations carried along with the *mecpQ*63<sup>∗</sup> allele, heterozygotes (*mecpQ*63∗/+) were outcrossed for at least 3 generations before to be incrossed to produce homozygote mutants (*mecpQ*63∗/*Q*63∗). Surprisingly, the homozygote *mecp2* mutant fish appears to develop normally, with no

**FIGURE 1 | Conserved synteny and generation of the zebrafish mecp2***Q***63<sup>∗</sup> mutation. (A)** Synteny analysis between zebrafish chromosome 8 and 11 and human chromosome X. The diagram shows regions of 2.07Mb and 0.07Mb, respectively, in zebrafish chromosome 8 and 11 and 0.5Mb in human chromosome X. The paralog hcfc1 group and ortholog to HCFC1 is indicated in blue, others in green. In addition, the second indicated paralog group is depicted in gray and mecp2 in red. The structure of these genomic regions shows that mecp2 is unique in the zebrafish genome and an ortholog of the human MECP2. **(B)** TILLING of ENU-mutagenized fish sperm after screening of exon 2 has allowed for the recovery of a C187T transition, creating a non-sense mutation. This mutation is upstream of the MBD and TRD functionnal domains, causing putatively a null mutation. **(C)** Sequence of mecp2 PCR from genomic DNA confirmed the C to T transition and the creation of a stop codon (underlined). **(D)** Western blot analysis of wild-type, mecp*Q*63∗/<sup>+</sup> and mecp*Q*63∗/*Q*63<sup>∗</sup> adult zebrafish brain tissue. Two quantities of proteins (10 and 5µg of total lysate) were loaded for each genotype. A major band around 75 kD reveals the presence of mecp2 in wild-type and mecp*Q*63∗/+, but not in mecp*Q*63∗/*Q*63<sup>∗</sup> adult zebrafish brain (minor bands might represent alternative post-translational mecp2 forms).

obvious morphological defects, they reach adulthood and reproduce. As maternally expressed *mecp2* mRNA has been detected in large amounts (Coverdale et al., 2004), *mecpQ*63∗/*Q*63<sup>∗</sup> fish were incrossed to remove any potential maternal contribution toward a milder phenotype. Similarly to the *mecpQ*63∗/*Q*63<sup>∗</sup> from *mecpQ*63∗/<sup>+</sup> parents, the progeny of *mecpQ*63∗/*Q*63<sup>∗</sup> fish develop apparently normally and are viable. Thus, maternal *mecp2* mRNA does not play a role in the moderation of the phenotype. However, albeit not quantified, in comparison to wild-type or *mecpQ*463∗/<sup>+</sup> fish, *mecpQ*63∗/*Q*63<sup>∗</sup> fish appear to have a slightly shorter lifespan. This lighter-than-expected phenotype in comparison to the mecp2-null mouse models could be explained by the fact that the mutation does not produce a null allele. Therefore, to assess how the mutation affects the protein level in the homozygote *mecp2* mutant, 5 and 10µg of adult brain proteins from wildtype, *mecpQ*63∗/<sup>+</sup> and *mecpQ*63∗/*Q*63<sup>∗</sup> fish were loaded on gels and subjected to Western blotting, with an antibody recognizing the C-terminus of the protein. In contrast to wild-type and heterozygote mutant fish, mecp2 protein could not be detected in *mecpQ*63∗/*Q*63<sup>∗</sup> fish (**Figure 1D**), indicating that the C187T transition is very likely to produce a null allele.

## **EARLY MOTOR BEHAVIOR**

Spontaneous contractions were monitored in 25 hpf dechorionated embryos at 21–22◦C. Wild-type embryos mainly showed unique side coiling of the trunk separated by a period of rest of several seconds. However, in some occasions, the contraction events led to several successive coilings without noticeable delay between each movement. Isolated, as well as multiple contraction movements were considered as single spontaneous motor activity events. The spontaneous motor activity of wild-type, heterozygote, and homozygote *mecp2* mutant embryos was then quantified. In line with a previous report (Pietri et al., 2009), wild-type embryos presented a median frequency of coiling of 5.12 events/min (*n* = 71) at 25 hpf (**Figure 2A**), not significantly different from *mecp2Q63*∗/<sup>+</sup> or *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos, with 5.66 events/min (*n* = 33) and 3.70 events/min (*n* = 33), respectively. Interestingly, the *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos showed higher activity in comparison to the others, with a larger number of contractions per event in contrast to the wild-type and *mecp2Q63*∗/<sup>+</sup> embryos. During the recording period, 80.6% of the *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos exhibit at least one event with more than one contraction, in contrast to 24.3% of the other embryos. Overall, a significant median increase was observed in *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos, where 30.55% of the events were composed of multiple coilings, in comparison to wild-type embryos (0%, *p* < 10−8, **Figure 2B**).

To further study the early development of motor behavior of the *mecp2Q63*∗/*Q63*<sup>∗</sup> fish, we characterized the escape response to tactile stimuli of 51 hpf embryos. In contrast to spontaneous activity or stimulus-evoked response in earlier embryos, where only the local circuit within the spinal cord is necessary and sufficient to produce motor behaviors (Pietri et al., 2009), escape-induced stimuli in 51 hpf embryos involves hindbrain structures, in particular the reticulospinal neurons (O'Malley et al., 1996; Liu and Fetcho, 1999). These cells notably receive inputs from the spinal sensory Rohon-Beard cells (Fetcho and Faber, 1988), and the multi-sensory recipient trigeminal ganglia afferents innervating the skin of the head (Kimmel et al., 1990). To test both pathways, we mechanically stimulated the trunk or the head of the embryos (see **Figure 2C** as an example of response to head tactile stimulation). Both types of stimulus produced an escape-like behavior, with embryos turning

away from the stimulus in a characteristic C-bend followed by a burst of swim bouts. **Figure 1C** shows an example of a 51 hpf *mecp2Q63*∗/*Q63*<sup>∗</sup> embryo where tactile stimulation of the head (0 ms) induced a C-bend (coiling period: 10–70 ms), and a swim bout (from 80 ms onward). In wild-type embryos, head and tail tactile stimulations produced behavioral responses with significantly different median C-bend durations [head (h, *n* = 11): 50 ms and tail (t, *<sup>n</sup>* <sup>=</sup> 20): 30 ms, *<sup>p</sup>* <sup>&</sup>lt; <sup>10</sup>−5, **Figure 2D**]. The same trend is observed between tail and head tactile stimulation responses in *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos [h (*<sup>n</sup>* <sup>=</sup> 16): 65 ms and t (*n* = 11): 40 ms, *p* = 0.0017], indicating that the principles governing both responses are conserved. Comparison of wild-type and *mecp*2*Q*63∗/*Q*63<sup>∗</sup> embryos for head or tail tactile stimulation

duration of tail as well as head-evoked coilings. Significant statistical differences are indicated by ∗ for *p* < 0.05 and ∗∗∗ for *p* < 0.001.

revealed however, for both types of stimuli, a longer duration of the C-bend for the *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos (h: 65 ms vs. 50 ms, *p* = 0.0113; and t: 40 ms vs. 30 ms, *p* = 0.0151).

## **SPONTANEOUS SWIMMING ACTIVITY**

Differences between wild-type and *mecp2* mutant fish were also assessed by monitoring their spontaneous motor behavior at 6 dpf. We found that the thigmotactic responses of *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae were different from their wild-type counterparts (**Figures 3**, **4**). Wild-type animals preferred to swim along the walls of the arena, while *mecp*2*Q*63∗/*Q*63<sup>∗</sup> larvae showed much less preference for the borders of the well, covering homogeneously most of its surface (**Figure 3A** for a single animal and for an average of *n* = 80, for both groups of larvae). We performed a quantitative analysis of the preferred swimming regions according to an inner region of radius 4.5 mm and an outer ring to the limit of the circular arena of radius 7.5 mm (**Figure 3B**). Wild-type animals spent 88.5 % of the time in the outer ring while *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae 73 % (*<sup>p</sup>* <sup>=</sup> <sup>0</sup>.001, **Figure 3C**), closer to the random case of 64% (dashed green line, **Figure 3C**). To discriminate whether this difference had a motor origin or was due to the different interactions with the wall, we analyzed their trajectories to and from the walls. In addition, we found that the distribution of the resting times of wild-type and *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae could be fitted to the same power law *<sup>Y</sup>* <sup>=</sup> *aX*−*k*, by adjusting the constant *<sup>k</sup>* accordingly. In comparison to wild-type, *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae showed a significantly lower median *k* constant (0.0525 vs. 0.2399 for wild-type larvae, *p* = 0.0007). In a reciprocal way, active time bouts had a larger *k*-value in *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae (2.79 vs. 1.92 for wild-type larvae, *p* = 0.0051). These altered values of *k* are coherent with the lower total motor activity (26.4 % vs. 56.0 % *p* < 10−6, **Figure 3D**), and lower bout velocities (13.0 mm/sec vs 18.9 mm/sec, *p* < 10−7, **Figure 3E**) for *mecp2Q63*∗/*Q63*<sup>∗</sup> in comparison to wild-type larvae. These results would then predict that *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae already at the outer ring would stay longer at this location, but we observed the opposite (**Figures 4A,B**). Analysis of the portion of the trajectories starting when the larvae reached the wall edge showed that most *mecp*2*Q*63∗/*Q*63<sup>∗</sup> larvae had a lower probability to stay in this region than their wild-type counterparts (**Figures 4A,B**). Interestingly, for the first 2 s, *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae had a higher probability of staying at the wall, consistent with the lower activity and velocities, but this quickly decreased afterwards to values below those of wild-type larvae, with significant differences after 5 s, reaching *p* < 10−<sup>10</sup> at 30 s. We also analyzed the portion of trajectories moving from the center toward the wall (using the inner/outer separation as *<sup>t</sup>* <sup>=</sup> 0) and found that *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae showed a significant lower probability in reaching the wall (after 30 s: *p* < 10−9, **Figure 4C**). To test if the latter is a consequence of the lower activity and velocity, we computed the length of the path from the center (within an area of less than 5% of the well) to the wall; a parameter completely independent of activity and velocity. *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae showed significantly longer paths than wild-type larvae (19.9 mm vs 12 mm, *p* < 10−13, **Figure 4D**). Overall, these results suggest that *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae have impaired locomotion and decreased thigmotaxis.

## **DISCUSSION**

In this study, we introduce the first mecp2-null RTT zebrafish model. In comparison to other mecp2-deficient animal models, this mutant shows a weaker phenotype as they are viable and

**FIGURE 3 | Kinematic analysis of free swimming wild-type and mecp***Q***63∗***/Q***63<sup>∗</sup> 6 dpf larvae. (A)** Example of traces for wild-type and mecp*Q*63∗/*Q*63<sup>∗</sup> larvae, obtained from a recording session of 15 min (top), and density maps representing the proportion of larvae at each position in the well (bottom, *n* = 80 for both groups of larvae). **(B)** The arena-surface division

into inner and outer regions as used for data analysis. **(C)** Quantification of the time spent in the inner and outer regions of **(B)**. Dotted green line is the random case. **(D)** Percentage of time when the larvae were active. **(E)** Mean velocity during activity. Significant statistical differences are indicated by ∗ for *p* < 0.05 and ∗∗∗ for *p* < 0.001.

fertile with a seemingly shorter lifespan. However, it shows clear behavioral impairments.

## **KNOCK-OUT OF mecp2 IN ZEBRAFISH PRODUCES AN UNEXPECTEDLY MILD PHENOTYPE**

In this model, suppression of the mecp2 functions was achieved by TILLING selection of a non-sense mutation upstream of the structural MBD domain. The position of the mutation within the gene predicted to be a null mutation. This prediction was supported by protein expression analysis that confirmed the complete loss of mecp2 in *mecp2Q63*∗/*Q63*<sup>∗</sup> zebrafish. In mouse, null mutations of MeCP2 induce phenotypes reminiscent of RTT (Chen et al., 2001; Guy et al., 2001). In particular, the lifespan of mutant mice is drastically reduced to 6 to 8 weeks after birth. To our surprise *mecp2Q63*∗/*Q63*<sup>∗</sup> zebrafish are viable and reproduce normally (in presence or absence of unaltered mecp2 maternal contribution). We noticed however, a tendency of this mutant fish to have a relatively shorter lifespan than wild-type zebrafish. This modest phenotype with respect to mice may indicate functional compensatory mechanisms, for instance, by the presence of a *mecp2* paralog gene. Synteny mapping, comparing human and zebrafish *mecp2* loci and extensive genome database mining of zebrafish and other teleost fish [in which the whole-genome duplication occurred before the radiation of these species (not shown; Postlethwait et al., 2004)] excludes the existence of *mecp2* paralog groups in the various fish species tested, indicating that the loss of the duplicated *mecp2* gene occurred probably early after the whole teleost genome duplication, and so in zebrafish. Partial functional compensation may also exist through the action of close MeCP2 protein family members, such as MBD2 that, like MeCP2 bears MBD and TRD domains. MBD2 is able to bind to methylated CpG dinucleotides and modulates the level of gene expression (Ng et al., 1999). This hypothesis has been raised to point out the lack of phenotype in null-MeCP2 mutant mouse in non-neural tissues, where MeCP2 is also expressed (Guy et al., 2001); this possibility has however not been thoroughly tested yet. In addition, due to the duplication of many genes in the zebrafish genome, compensatory mechanisms can take place at the level of the *mecp2* target genes. For instance, MeCP2-deficient mice present various alterations of NMDA receptor expressions that are region and age specific (Blue et al., 2011). In zebrafish, all five NMDA receptors present in mammals are duplicated (Cox et al., 2005), raising the possibility that *mecp2*-induced alterations of the expression of one of the genes in a paralog group is compensated by expression of the second one. Moreover, these receptors are heteromeric, and if compensation within a paralog group cannot be achieved, alternative forms of the receptors could, at least partially, be used. Finally, the difference in the phenotypes between mouse and zebrafish might be explained by the far greater regenerative abilities of the zebrafish brain than those of the mammalian ones. In contrast to mammals, zebrafish neurogenesis occurs throughout life in many brain regions (Zupanc, 2008). While new-born neurons integrate into already established networks, "old" neurons are eliminated by apoptosis. The late onset of the neural phenotype of the MeCP2-deficient mouse (Chen et al., 2001; Guy et al., 2001) and postnatal neural restoration of MeCP2 expression in MeCP2-null mouse were shown to be sufficient to largely rescue the RTT phenotypes (Guy et al., 2007), thus, suggesting that alteration of MeCP2 functions perturbs the maintenance of the nervous system rather than its initial development. It is conceivable that the constant addition of new neurons in zebrafish neural circuits could prevent the appearance of the lethal phenotype seen in MeCP2-null mouse models. These various compensatory mechanisms that are for some of them uniquely present in zebrafish, may explain its high resilience to the loss of *mecp2*. On the other hand, the ENU-mutagenesis used in the TILLING procedure produces a large number of mutations. Some may be carried along with the mutation of interest, even after several generations. We thus cannot completely exclude the presence of a second genetically-linked mutation to *mecp2Q63*∗. Our mecp2-null model presents a milder-than-expected phenotype. Thus, if indeed zebrafish and mammals mecp2 functions are conserved, a second genetic alteration would have to be a suppressor of the effect of the *mecp2Q63*<sup>∗</sup> mutation. The probability of such mutation in this context is very low. In-depth sequencing or s*ingle nucleotide polymorphisms* analysis of the mecp2 region could be used to rule out this concern.

## **SPONTANEOUS AND EVOKED EARLY MOTOR ALTERATIONS SUGGEST AN EXCITATION/INHIBITION UNBALANCE**

By 24 hpf, spontaneous motor activity seems to be triggered by an intraspinal premotor circuit (Saint-Amant and Drapeau, 1998; Pietri et al., 2009). At this developmental stage, the excitatory components rely on electrical coupling via gap junctions, reinforced by nascent glutamatergic synapses (Saint-Amant and Drapeau, 1998; Pietri et al., 2009). On the other hand, inhibition appears to be driven only by glycinergic inputs, as evidence of GABAergic participation in the early spontaneous activity could not be detected (Saint-Amant and Drapeau, 2000, 2001). The similarity in the frequency of spontaneous muscular contractions between wild-type and mutant mecp2 embryos suggests that excitation in the spinal cord may remain intact in *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos, while the sustained spontaneous motor activity events in these embryos would argue for a decrease in inhibition efficacy, likely through perturbation of the glycinergic system. A less parsimonious hypothesis would be that both excitation and inhibition are depressed in 25 hpf *mecp*2*Q*63∗/*Q*63<sup>∗</sup> embryos, but with a stronger alteration of the glycinergic inhibitory component. This hypothesis is supported by the observed tendency for a lower rate of spontaneous activity events in 25 hpf *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos. In contradiction with these hypothesis, one study on the medulla of MeCP2-null mouse at postnatal day 7 has shown that glycinergic transmission is unaltered, while GABAergic transmission is strongly depressed (Medrihan et al., 2008). However, modulation of the defects of particular neurotransmitters appears to be region and age specific (Blue et al., 2011). It is therefore plausible that alteration of glycinergic transmission is either transient or only effective in the spinal cord of *mecp2Q63*∗/*Q63*<sup>∗</sup> fish.

One of the symptoms observed in autistic patients, including RTT patients, is their hyper-responsiveness to sensory stimuli (Belmonte et al., 2004). To investigate sensorimotor behavior in zebrafish early development, light mechanical stimuli were thus applied on the trunk or the head of the embryos to induce a startle/escape behavior. By 2 dpf, head and tail sensory stimuli are integrated through hindbrain reticulospinal neurons to produce a motor response. These sets of neurons receive inputs from the sensory trigeminal and Rohon-Beard neurons innervating respectively the head and body skin (Kimmel et al., 1990; Kuwada et al., 1990). While they appear similar at 21 hpf (Saint-Amant and Drapeau, 1998), responses to head and tail tactile stimulations showed different kinematics in 4–5 dpf larvae (Liu and Fetcho, 1999; Liu et al., 2012), reflecting the involvement of different reticulospinal neurons in mediating the escape response (Gahtan et al., 2002). In 51 hpf embryos, tail tactile stimulations induced coiling responses of shorter durations than those evoked by head stimulations, similar to what was found at 5 dpf, and thus suggesting that t different sets of reticulospinal neurons are already required at an early stage of development. *Mecp2Q63*∗/*Q63*<sup>∗</sup> embryos responded to tactile stimulations showing a similar difference between the duration of head and tail-induced responses seen in wild-type embryos. This suggests that the structure of the network necessary for both sensory-evoked motor responses is not altered. However, careful examinations of the mouse models, previously believed to be asymptomatic in their early development, have uncovered alterations of their sensory reflexes and sensory-motor gating development (Picker et al., 2006; Santos et al., 2007). Similarly, at 51 hpf, *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos showed alterations of sensory-evoked motor responses characterized by a lengthening of the coiling duration. Both trigeminal and Rohon-Beard sensory neurons implicated in the transmission of the mechanical stimulation as well as the Mauthner neurons are glutamatergic neurons (Higashijima et al., 2004). At this age, glycinergic neurotransmition appears to remain the main inhibitory system in the spinal cord (Saint-Amant, 2010). Similar alteration of the coiling responses of both sensory stimulations in *mecp2Q63*∗/*Q63*<sup>∗</sup> embryos may imply that only the premotor network (downstream of the reticulospinal neurons) is affected and like in earlier stage embryos, the perturbation of the glycinergic system is responsible for the observed phenotype. Alternatively, glutamatergic neurotransmission may be altered in a similar way in both types of sensory neurons. This hypothesis can not be ruled out, as our experiments did not allow for the quantification of the delay of the response to a tactile stimulation, which would be indicative of such perturbation. It is therefore possible that defects in glutamatergic excitation are combined with alterations of the inhibitory neurotransmission, as seen in the mouse model of RTT (Chao et al., 2007, 2010; Medrihan et al., 2008; Blue et al., 2011).

## **Mecp2-MUTANT LARVAE SHOWED REDUCTION IN ACTIVITY AND A DECREASE IN ANXIETY-LIKE BEHAVIOR**

Patients with RTT suffer from pronounced alterations of their motor systems. These defects have been phenocopied in RTT mouse models. We therefore tested our zebrafish model for motor dysfunctions in 6 dpf larvae in the open-field paradigm. As seen in mouse models, alteration of *mecp2* functions in zebrafish larvae induced a sharp decrease of the activity level, notably characterized by an increase of the resting times between swimming bouts. Interestingly, both resting times in wild-type and *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae followed the same power-law distribution. The fact that a power law can describe the kinematics of both *mecp2Q63*∗/*Q63*<sup>∗</sup> and wild-type larvae suggests that the mutation modulates wild-type neural networks rather than generating major muscular or/and morphological abnormalities that could indirectly affect motor behavior. (Harnos et al., 2000; Faure et al., 2003). In mecp2-null mouse models, the different aminergic systems have been shown to be specifically defective (Panayotis et al., 2011). In particular, defects in the dopaminergic system have been shown to be responsible for the movement abnormalities (Samaco et al., 2009; Gantz et al., 2011). In zebrafish, dopaminergic systems regulate the development of locomotor circuits, putatively by regulating synaptogenesis or influencing other neuromodulatory pathways (Thirumalai and Cline, 2008). It is thus possible that suppression of *mecp2* functions in zebrafish larvae induces a perturbation of neuromodulation of the motor network development that would then underlie the motor phenotype.

The dopaminergic, among other neuromodulatory systems, is also implicated in the control of anxiety-related behaviors (Wood and Toth, 2001). Thus, we tested for thigmotaxis in 6 dpf larvae, a behavioral test commonly used to evaluate the level of anxiety in humans and other animals. In zebrafish, this test has been particularly used in adults (Peitsaro et al., 2003; Anichtchik et al., 2004), but recently validated in larvae as young as 5 dpf (Colwill and Creton, 2011; Schnorr et al., 2012). In larvae, as well as in adults, thigmotaxis has been interpreted as a defensive strategy to avoid detection by predators and is reduced in familiar environments (Colwill and Creton, 2011) or after exposure to anxiolytic drugs (Schnorr et al., 2012). Studies of anxiety in mouse models of RTT have brought so far inconsistent results on the presence of elevated or reduced anxiety-like behaviors. The complexity of this behavior involving several brain structures, neurotransmitter systems and hormonal neuromodulation makes the interpretation of the various tests difficult and may thus explain such discrepancy. However, place preference analysis in an open-field arena, which would be the equivalent of wells for zebrafish, have shown an increase in anxiety levels in 3 and 5 month old MeCP2<sup>308</sup> mice (Shahbazian et al., 2002b) and in 8-week old mecp2-null mice (Stearns et al., 2007). This is in contradiction with our results showing that *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae spent less time close to the well's edge than wild-type larvae. However, motor dysfunction present in the mutant larvae may lead to false interpretation of the place-preference results. Thus, a subset of trajectories were chosen to test for attraction toward edges and maintenance of edge preference during the active phases of swimming. *mecp2Q63*∗/*Q63*<sup>∗</sup> larvae showed a clear reduction in both parameters, strongly suggesting a decreased level of thigmotaxis in comparison to wildtype age-matched larvae. Moreover, the mutant larvae covered more distance in the inner region of the well than their wildtype counterparts in their periods of activity, a variable that has also been shown to be indicative of the level of anxiety in mice

## **REFERENCES**

behavioural changes in zebrafish *Danio rerio* after systemic administration of 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine. *J. Neurochem*. 88, 443–453. doi: 10.1111/j.1471- 4159.2004.02190.x

Ballas, N., Lioy, D. T., Grunseich, C., and Mandel, G. (2009). Non-cell autonomous influence of MeCP2 deficient glia on neuronal dendritic in the open-field paradigm (Paylor et al., 1998). Overall, these results suggest that *mecpQ*63∗/*Q*63<sup>∗</sup> larvae are less anxious than their wild-type counterparts.

Several autistic features have also been explained as a possible dysfunction of the reward system (Scott-Van Zeeland et al., 2010; Dichter et al., 2012). Interestingly, the dopaminergic system is also a core component of the reward circuits (Dichter et al., 2012). The decrease in locomotor activity of *mecpQ*63∗/*Q*63<sup>∗</sup> larvae could thus be explained as a reduction of interest in environment exploration. The decreased thigmotaxis could be the result of avoiding tactile stimulation from the wall, as expected from hyper-responsive autistic patients (Belmonte et al., 2004).

## **CONCLUSION**

While rodents have been the historical model of choice to study the genetic alterations present in neurodevelopmental and neurodegenerative human diseases, zebrafish is currently becoming an important complementary model for translational neuroscience research, as it is the only suitable vertebrate model for high throughput drug screening. Analysis of *mecp2*-deficient zebrafish during early development has uncovered motor defects compatible with motor phenotypes observed in MeCP2-null mouse models and RTT patients. The zebrafish RTT model, in contrast to other vertebrate ones, will enable monitoring brain dynamics, neural morphology and behaviors, through the entire normal lifespan of the organism. Furthermore, the non-lethality of this model will enable the study of the natural genetic compensatory mechanisms. Deciphering these mechanisms would potentially lead to alternative directions in the understanding of the pathophysiology of RTT and open the way for the discovery of novel treatments.

## **ACKNOWLEDGMENTS**

The authors thank our respective teams for their support during the elaboration of this work, in particular Adrien Jouary and Morgane Nouvian for fruitful discussions, the animal facility staff of the University of Oregon and the Institut de Biologie de l'Ecole Normale Supérieure for the constant care of the zebrafish and Dr. J. LaSalle for the anti-MECP2 antibody. This research was supported by fellowships from Autism Speak and the Fondation pour la Recherche Medicale to Thomas Pietri, and Juan de la Cierva (Spain) to Angel-Carlos Roman, grants from NINDS R01 NS065795 to Philip Washbourne, NIH R01 HD076585 to Cecilia B. Moens, Ministerio de Economia as Plan Nacional (Spain), EraSysBio+ ZeBrain to Gonzalo G. de Polavieja and Germán Sumbre and ERC stg 243106 to Germán Sumbre.

morphology. *Nat. Neurosci*. 12, 311–317. doi: 10.1038/nn.2275

Beckel-Mitchener, A., et al. (2004). Autism as a disorder of neural information processing: directions for research and targets for therapy. *Mol. Psychiatry* 9, 646–663.

Blue, M. E., Kaufmann, W. E., Bressler, J., Eyring, C., O'Driscoll, C., Naidu, S., et al. (2011). Temporal and regional alterations in NMDA receptor expression in MeCP2-null

*Sci. U.S.A*. 102, 12560–12565. doi: 10.1073/pnas.0506071102

1035–1046. doi: 10.1016/S0896- 6273(01)00416-0

active genes. *Proc. Natl. Acad. Sci. U.S.A*. 104, 19416–19421. doi: 10.1073/pnas.0707442104

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 12 April 2013; accepted: 21 June 2013; published online: 16 July 2013.*

*Citation: Pietri T, Roman A-C, Guyon N, Romano SA, Washbourne P, Moens CB, de Polavieja GG and Sumbre G (2013) The first mecp2-null zebrafish model shows altered motor behaviors. Front. Neural Circuits 7:118. doi: 10.3389/fncir. 2013.00118*

*Copyright © 2013 Pietri, Roman, Guyon, Romano, Washbourne, Moens, de Polavieja and Sumbre. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

## The medial habenula as a regulator of anxiety in adult zebrafish

## *Ajay S. Mathuru1 and Suresh Jesuthasan1,2,3\**

*<sup>1</sup> Neural Circuitry and Behavior Laboratory, Institute of Molecular and Cell Biology, Singapore, Singapore*

*<sup>2</sup> Neuroscience and Behavioral Disorders Program, Duke-NUS Graduate Medical School, Singapore, Singapore*

*<sup>3</sup> Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore*

*\*Correspondence<EMAIL_ADDRESS>
#### *Edited by:*

*Gonzalo G. De Polavieja, Instituto Cajal. CSIC, Spain*

#### *Reviewed by:*

*Hitoshi Okamoto, RIKEN Brain Science Institute, Japan*

The habenula consists of a set of nuclei located in the epithalamus. It regulates the release of multiple neuromodulators including serotonin and dopamine, and consists of two major subdivisions medial and lateral. In all vertebrates, the medial habenula projects to the interpeduncular nucleus (IPN), a midline structure with poorly defined functions (Morley, 1986). Both the medial habenula and the IPN are rich in nicotinic receptors (nAChR). Activity in this pathway, triggered by opioids and nicotine, leads to a rise in dopamine in the nucleus accumbens (Glick et al., 2006; McCallum et al., 2012) and thus underlies the rewarding aspect of substance abuse. Strong activation of nicotinic receptors in the medial habenula or IPN, however, is sufficient to mediate the aversion to high concentration of nicotine (Fowler et al., 2011; Frahm et al., 2011). In contrast, absence of activity in this pathway is critical for the effects of withdrawal (Salas et al., 2009; Baldwin et al., 2011). Hence, depending on the level of activity, the medial habenula-IPN pathway can trigger reward, aversion or the physical and emotional changes that are characteristic of withdrawal.

The medial habenula regulates the expression of fear in zebrafish. Following silencing with tetanus toxin or lesioning with nitroreductase (Agetsuma et al., 2010; Lee et al., 2010), enhanced freezing was seen in aversive conditioning paradigms in both adult and larvae. The freezing response in habenula-lesioned fish was experience-dependent. In the case of adults, both control and lesioned fish froze when first exposed to an electric shock paired with a red light (the conditioning stimulus, CS) in a Pavlovian conditioning paradigm. As more shocks were delivered, freezing decreased in control fish, but not in habenula-lesioned fish (Agetsuma et al., 2010). The experiment with larval fish involved instrumental conditioning: fish had to swim away from the side of the tank containing a red light, which had been paired with a mild electric shock. In this case, freezing appeared gradually in lesioned fish in the later half of the conditioning session, but never in control fish (Lee et al., 2010).

One interpretation that has been suggested for these observations is that silencing the medial habenula biases fish to a passive coping strategy of freezing, due to modulation in the activity of the downstream griseum centrale [homologous to periaqueductal gray (PAG) in mammals] and nucleus incertus (Okamoto et al., 2012). As different regions of the PAG have been implicated in selection of different actions for coping against stress, a role for the medial habenula in such selection via PAG is a distinct possibility. An additional possibility is that medial habenula silencing increases anxiety in animals.

A consequence of elevated generalized anxiety in an animal is predisposition toward larger fear responses (Davis et al., 2010). The swimming behavior of an isolated fish when introduced into a novel tank can be used as a measure of baseline generalized anxiety (Levin et al., 2007): anxious fish spend more time near the bottom in a novel tank. Therefore, by using this assay, it is possible to quantify if adult fish where the medial habenula has been silenced display a similar, a lower, or a higher level of baseline anxiety when compared to control fish. We compared the behavior of adult fish that express the light chain of tetanus toxin (TeTXlc) in the medial habenula (Lee et al., 2010), under the control of the GAL4s1019t driver (Scott et al., 2007), with fish that did not express this transgene. TeTXlc expressing fish spent more time in the bottom half of the tank compared to controls (**Figures 1A**,**B**), consistent with the possibility that these fish may indeed have a higher baseline generalized anxiety.

Mild stressors can elicit disproportionate reactions in anxious animals. In rats, for example, strong illumination, which is anxiogenic, causes an abnormally large startle to a tone (Walker and Davis, 1997). We examined the response of TeTXlc-expressing fish to naturalistic stimuli that are expected to be mildly stressful. Low concentration of the alarm substance (Jesuthasan and Mathuru, 2008) normally triggers a response that naïve, control fish recover from within seconds (0.1 unit; Mathuru et al., 2012). We followed this with an overhead shadow, which is thought to mimic the threat of a predator (Luca and Gerlai, 2012). These stimuli provide an opportunity to examine the role of medial habenula when the subject is challenged with stimuli that mimic complex natural stressors as opposed to its role in conditioning experiments that use unnatural repetitive stimuli. In such conditions, control fish displayed behavior indicative of mild fear—marginal increase in episodes of darting [Wilcoxon Signed Rank Test (*n* = 7), *p* = 0.1; **Figure 1F**], while other measures remained unchanged. TeTxlcexpressing fish, in contrast, spent more time spent in the bottom quarter of the tank (Mann–Whitney *U*-test, n1, n2, 7, 9, *U* = 7, *p* = 0.005; **Figure 1C**), displayed

**FIGURE 1 | (A,B)** Novel tank assay. Area in each color shows percentage time spent in either half of a novel tank in one minute by five Control fish and five TeTXlc expressing fish. Arrows indicate median swim time in the bottom half for controls (20.4 ± 3.7%) and TeTXlc fish (48.6 ± 9%). **(C–F)** Response to dilute alarm substance and an overhead shadow. **(C)** Time spent in the bottom quarter of a tank, and median number of **(D)** slow swimming, **(E)** pauses, and **(F)** darts. Non-parametric, Mann–Whitney *U*-test was used to compare behavior of control and TeTXlc fish following exposure to the stimuli. (Mann–Whitney *U*-test, ∗*p* < 0.05, ∗∗*p* < 0.01, ∗∗∗*p* < 0.001). Zebrafish expressing TeTXlc-CFP in the habenula were generated by crossing GAL4s1019t with UAS:TeTXlc-CFP, and selected on the basis of CFP fluorescence in the brain. For the novel tank assay, fish were transferred into

a glass tank measuring 20 × 12 × 5 cm—L × H × W containing 1 L of water and observed for 1 min. Digital videos were recorded as described before (Mathuru et al., 2012). Fish position in the tank was tracked and analyzed automatically using "track objects" algorithm in MetaMorph. 6.3 and custom written macros for Excel. Alarm substance was prepared as described before (Mathuru et al., 2012). This was introduced into the tank and a shadow, created by passing a hand under the overhead light, was presented after 2 min. Darting was defined as episodes during which the swimming speed exceeded baseline speed by more than 8 SD, slow swim episodes were defined as episodes greater than one second in duration during which the swim speed never exceeded half the mean baseline speed, while one second of immobility (speed less than 3 mm/s) was considered as a pause episode.

more episodes of slow swimming (Mann– Whitney *U*-test, n1, n2, 7, 9, *U* = 3, *p* = 0.001; **Figure 1D**) and pauses (Mann– Whitney *U*-test, n1, n2, 7, 9, *U* = 14, *p* = 0.021; **Figure 1E**). Darting episodes were comparable to controls (MW n1, n2, 7, 9, *U* = 17.5, *p* = 0.435; **Figure 1F**). The response of TeTxlc-expressing fish resembled that of naïve fish that exhibit intense innate fear upon exposure to a high concentration of the alarm substance (1 unit; Mathuru et al., 2012). In other words, TeTxlc-expressing fish show a disproportionate response when presented with multiple mildly stressful stimuli. Notably, TeTxlc-expressing fish display both active and passive responses, suggesting that there is an increase in the intensity of fear expression rather than a bias toward a particular type of action.

By examining responses to a novel environment, as well as to the alarm substance along with an overhead shadow, i.e., conditions where experience is absent, the behavioral responses of fish with dysfunctional medial habenula appear to be consistent with that of animals in a state of elevated baseline anxiety. Behavioral deficits are manifest primarily in the presence of stressors, similar to the effects of nicotine withdrawal in rats (Jonkman et al., 2008).

The mechanism by which the habenula regulates a response to stress, both experience dependent and independent is likely to be complex, given the multiplicity of targets and modulators in the medial habenula-IPN pathway, which include somatostatin (Morley et al., 1985), met-Enkephalin, and substance P (Hamill et al., 1984). It is possible that regulation of serotonergic neurons in the raphe by the IPN also plays a role in modulating the response to aversive stimuli. Another intriguing hypothesis that has been suggested recently is that the absence of nicotine in addicts causes an increase in activity in the lateral habenula, which then leads to a decrease in dopamine release from the ventral tegmental area (VTA), thereby causing symptoms associated with withdrawal that include increased anxiety (Baldwin et al., 2011).

Is it possible then that silencing the medial habenula has a similar outcome, i.e., leads to an increase of activity in the lateral habenula? Consistent with the notion that the medial habenula can regulate lateral habenula outputs, projections from the medial to the lateral habenula have been detected in the rat (Kim and Chang, 2005), although it is not known if these projections are inhibitory. As the lateral habenula is able to use errors in prediction (based on experience) to trigger a change of state (Matsumoto and Hikosaka, 2007; Hong and Hikosaka, 2008), this region may also be involved in mediating the experience-dependent deficits of silencing the medial habenula. If activity in an inhibitory pathway from the medial habenula to the lateral habenula is reduced, the overall inhibitory tone on lateral habenula will also be reduced. In this scenario, transmission via the lateral habenula would be increased, and the outcome would be similar to that seen in rats where such an increase is associated with a predisposition for helpless behavior (Li et al., 2011), which includes increased freezing.

Thus, one step to understand both nicotine withdrawal and anxiety will be to characterize signaling between medial and lateral subnuclei of the habenula, in addition to examining downstream circuits. These subnuclei have remained in close proximity throughout vertebrate evolution, and it is possible that intra-habenula signaling modulates output, eventually effecting anxiety and motivated behaviors in animals.

## **REFERENCES**

*Received: 27 February 2013; accepted: 03 May 2013; published online: 274 May 2013.*

*Citation: Mathuru AS and Jesuthasan S (2013) The medial habenula as a regulator of anxiety in adult zebrafish. Front. Neural Circuits 7:99. doi: 10.3389/ fncir.2013.00099*