IntroductionNeurons have the amazing ability to self-assemble into highlyorganized circuits. These circuits give rise to our perceptions,thoughts and emotions, and determine how we experience ourworld. Disorders in neural development, therefore, can oftencompromise the quality of life. To date, there are no cures forprevalent neurodevelopmental disorders such as autism, epilepsyand schizophrenia, and there are many gaps in what is known aboutthe underlying causes of these conditions. Animal models that allowfor a disorder to be studied at multiple levels, from molecules tobehavior, can provide a more complete understanding of theassociated gene locus and underlying mechanism(s), therebypromoting the design of novel approaches for treatment andprevention.

The Xenopus laevis tadpole possesses many qualities that makeit a powerful model to study disorders of the developing nervoussystem. First and foremost, essentially every stage of normal neuraldevelopment, from neurogenesis and differentiation to axonpathfinding, synapse maturation and circuit refinement, has beenstudied in detail in Xenopus tadpoles (Cline and Kelly, 2012; Saneset al., 2012). Such a detailed understanding of normaldevelopmental processes is invaluable when seeking to determinehow they can malfunction. Compared with mammalian neuralcircuits, those of the tadpole are simpler, yet homologous in theirbasic organization. For example, in the tadpole retinotectal circuit(Fig. 1), retinal ganglion cells (RGCs) in the eye project their axonsto the brain, where they synapse onto tectal neurons in the

contralateral optic tectum (Gaze, 1958; Sperry, 1963), a midbrainstructure that is homologous to the mammalian superior colliculus.The RGC axons form a highly organized topographic map withintheir target structure, with neighboring RGCs making synapsesonto neighboring tectal neurons. This mirrors what is observed inmany mammalian sensory circuits, including those within thehuman nervous system. Furthermore, as in most mammalianexcitatory synapses, RGC axons release glutamate, and tectalneurons express AMPA and NMDA glutamate receptors (Wu etal., 1996). Building on the contributions of the Xenopus model tothe field of embryology, many aspects of Xenopus neural circuitdevelopment have been carefully studied, and meticulouslydescribed across the key developmental stages. For instance, it iswell established that the axons of the RGCs reach the tectum ataround developmental stage 39 [4-5 days post-fertilization (dpf)](Holt, 1989; Dingwell et al., 2000), that the most dynamic phase ofcircuit formation – both morphologically and functionally – occursbetween stage 44 and 47 (7-10 dpf), and that by stage 49 (~16-24dpf) the circuit becomes more refined and stable (Sakaguchi et al.,1984; Cline et al., 1996b; Pratt and Aizenman, 2007).

Experimentally, Xenopus tadpoles pose many advantages. Becauseof their transparency, and the extreme dorsal location of the brain,the axons and dendrites of single living neurons can be imaged invivo (Harris et al., 1987; Ruthazer et al., 2003; Cohen-Cory, 2007; Liet al., 2011; Dong and Aizenman, 2012) and, better yet, in awakeanimals (Chen et al., 2012; Hossain et al., 2012). Time-lapse imagingof radial glia in the tectum has provided the first in vivo descriptionof how neural activity affects the structure and function of these cells(Tremblay et al., 2009), and advances in morphometric software (Liuet al., 2009; Chen et al., 2012) have allowed for an improved abilityto track and measure all processes in 3D across short intervals overlong periods of time (Hossain et al., 2012). Furthermore, calciumimaging in tadpoles can be carried out readily (Tao et al., 2001; Juneket al., 2010; Xu et al., 2011), including in vivo recordings from intact

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1University of Wyoming, 1000 E University Avenue, Laramie, WY 82071, USA2Brown University, 45 Prospect Street, Providence, RI 02912, USA*Author for correspondence (kpratt4@uwyo.edu)

© 2013. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distributionand reproduction in any medium provided that the original work is properly attributed.

The Xenopus tadpole model offers many advantages for studying the molecular, cellular and network mechanismsunderlying neurodevelopmental disorders. Essentially every stage of normal neural circuit development, from axonoutgrowth and guidance to activity-dependent homeostasis and refinement, has been studied in the frog tadpole,making it an ideal model to determine what happens when any of these stages are compromised. Recently, thetadpole model has been used to explore the mechanisms of epilepsy and autism, and there is mounting evidence tosuggest that diseases of the nervous system involve deficits in the most fundamental aspects of nervous systemfunction and development. In this Review, we provide an update on how tadpole models are being used to study threedistinct types of neurodevelopmental disorders: diseases caused by exposure to environmental toxicants, epilepsy andseizure disorders, and autism.

Modeling human neurodevelopmental disorders inthe Xenopus tadpole: from mechanisms totherapeutic targetsKara G. Pratt1,* and Arseny S. Khakhalin2

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awake animals (Chen et al., 2012; Podgorski et al., 2012; Imaizumiet al., 2013). Expression of genes of interest can also be achieved invivo, using electroporation-based protocols (Haas et al., 2001; Haaset al., 2002; Bestman et al., 2006), or by mRNA injection intoappropriate cells of the early embryo (Demarque and Spitzer, 2010).Because of the relatively high permeability of the tadpole blood-brainbarrier, pharmacological manipulations of the nervous system areusually achieved by simply adding the pharmacological agent to thetadpole rearing solution. Electrophysiological techniques have beensuccessfully employed in Xenopus to quantify network connectivity

(Pratt and Aizenman, 2007; Li et al., 2009; Pratt and Aizenman, 2009;Straka and Simmers, 2012), synaptic maturation (Wu et al., 1996;Akerman and Cline, 2006; Aizenman and Cline, 2007; Deeg et al.,2009; Khakhalin and Aizenman, 2012), synaptic plasticity (Engertet al., 2002; Mu and Poo, 2006; Pratt et al., 2008; Tsui et al., 2010)and cell intrinsic properties (Aizenman et al., 2003; Pratt andAizenman, 2007; Winlove and Roberts, 2011). The behaviorscontrolled by corresponding neural circuits, including several typesof escape behaviors (Roberts et al., 2000; Wassersug and Yamashita,2002; Dong et al., 2009; Sillar and Robertson, 2009), orienting

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Fig. 1. The Xenopus tadpole as a research model, shown with key experimental techniques that are used to differentiate between normal and abnormalpatterns of neural development. (1) Top: view of the animal at ca. 3 weeks post-fertilization. Several behavioral tests can be used to assess brain development:for example, wild-type animals usually swim along the sides of the container (represented by a circle; bottom), whereas animals with altered excitation/inhibitionbalance tend to circle in the middle of it. (2) Top: general view of the brain. OB, olfactory bulbs; OT, optic tectum; HB, hindbrain: SC, spinal cord; red, projectionsfrom the retina; green, tectal projections to the hindbrain; blue, descending projections to the spinal cord. An isolated brain provides an accessible in vitro

preparation, and whole-brain immunostaining (bottom) can be used to quantify global alterations in brain biochemistry (an exaggerated staining for GABA isshown). (3) Top: horizontal section of the optic tectum (OT) and caudal forebrain (FB); at this level, Ca2+ imaging can be used to detect abnormal seizure-likepatterns of activity (bottom). (4) At the neuron level, in vivo or ex vivo imaging allows assessment of cell morphology development. (5) At the synaptic level,electrophysiology offers a way to quantify maturation of synaptic and intrinsic properties of the cell through recordings of (a) evoked synaptic responses, (b)spiking in response to current injections and (c) spontaneous synaptic activity. The figure is inspired by experimental data published in the following papers:(Aizenman et al., 2002; Bestman et al., 2006; Ruthazer et al., 2006; Pratt and Aizenman, 2007; Hewapathirane et al., 2008; Bollmann and Engert, 2009; Hiramotoand Cline, 2009; Straka, 2010; Bell et al., 2011; Marshak et al., 2012; Miraucourt et al., 2012).

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reflexes (Pronych et al., 1996; Simmons et al., 2004; Straka, 2010)and social behaviors (Katz et al., 1981; Villinger and Waldman, 2012),have been well described, and can be experimentally manipulated(Lum et al., 1982; Jamieson and Roberts, 2000; Wassersug andYamashita, 2002; Simmons et al., 2004; Dong et al., 2009; Straka,2010). To sum up, these experimental approaches enable developingneural circuits to be examined at the molecular, cellular andbehavioral levels – all in the same organism (Fig.  1). Moreover,humans are genetically closer to Xenopus than to similar modelorganisms, such as zebrafish, because teleosts (the ray-finned fishes)are known to have divergent and highly specialized genomes(Postlethwait et al., 2004; Nakatani et al., 2007; Rash et al., 2012).Combined with the relatively low cost of housing, large number ofembryos generated from one mating, and the ease of embryologic(De Robertis, 2006; Harland and Grainger, 2011; Pai et al., 2012)and surgical (Constantine-Paton and Capranica, 1976; Filoni, 2009;McKeown et al., 2013; Elliott et al., 2013) manipulations, thesequalities render Xenopus an ideal model for neurodevelopmentalresearch.

In this Review, we first highlight how tadpoles have been usedas a model for assaying the effects of environmental chemicals onneurodevelopment, and how the model itself has evolved and beenrefined over the years. We then describe a recently designed tadpolemodel of epileptic seizures that has already led to the finding of abuilt-in protective mechanism that is activated in response to aseizure. Finally, we present a new and exciting tadpole model tostudy autism.

Characterizing the effects of environmental toxicantson development in a tadpole modelDuring development, neural circuits can be particularly sensitiveto chemicals in the environment. In humans, for example, dosesof methylmercury that are neurotoxic to the embryonic centralnervous system (CNS) have no effect on the maternal CNS(Castoldi et al., 2001). Similarly, exposure of the developing nervoussystem of a rat pup to lead (a heavy metal) often results inencephalopathy, whereas the mature rat brain remains unaffectedwhen exposed to the same amount of lead (Holtzman and Hsu,1976). Thus, chemicals in the environment that are deemed to beinnocuous to the adult CNS can be harmful to a developing brain.

Having been used for decades by researchers in academia aswell as the US government’s Environmental Protection Agency(EPA) to assay toxic and teratogenic effects of environmentalchemicals, the Xenopus tadpole is not new to the field ofembryotoxicology (Dumpert and Zietz, 1984; Degitz et al., 2003;Richards and Cole, 2006; Berg et al., 2009). The tadpole has servedas a workhorse for these studies mostly because theirmetamorphosis from tadpole to frog depends entirely on thyroidhormone (TH) (Damjanovski et al., 2000), and, in turn, one of themost prevalent environmental contaminants are the TH inhibitors,a major class of endocrine disruptors. In the tadpole, if TH actionis inhibited, metamorphosis stalls, whereas exposure to TH in pre-metamorphic tadpoles induces precocious metamorphosis(Helbing et al., 2007). Because the progression of metamorphosisis well described and obvious, alterations can be readily identified.Hence, this became a convenient way to test many classes ofchemicals for their ability to disrupt TH activity (Gutleb et al., 2000;Tietge et al., 2005; Cheng et al., 2011; Lorenz et al., 2011). One of

the major targets of TH, however, is the brain, where disruptionof normal TH activity can lead to neurodevelopmental defects(Zoeller and Crofton, 2000). Given that it is unlikely that the moresubtle defects associated with neurodevelopment (such asincomplete synapse refinement for example) would disrupt therelatively gross changes associated with metamorphosis (such asloss of the tail, emergence of limb buds and formation of lungs),disorders in nervous system development could go undetected.Thus, it was necessary to develop a more sensitive molecularapproach for the identification of neural-specific molecularmarkers that are associated with TH disruption. Experiments usingquantitative reverse transcriptase PCR (qRT-PCR) revealed thatthe TH inhibitors methimazole and perchlorate alter neural THreceptor expression in brain tissue of stage-54 tadpoles (Zhang etal., 2006). Furthermore, in a detailed study combining cDNA arrayanalysis and qRT-PCR, perchlorate was found to significantlyincrease the expression of several neural mRNAs (Helbing et al.,2007), including the mRNA for β-amyloid precursor protein, aprotein whose improper processing has been highly implicated inAlzheimer’s disease, and mRNAs that encode for myelin basicprotein and myelin proteolipid protein, both of which are majorcomponents of the myelin sheath that insulates axons andfacilitates appropriate action potential conduction. The effects thatthese perchlorate-induced increases in mRNAs could have on thedeveloping tadpole brain, however, remain unknown.

Advances in both imaging and electrophysiological approacheshave enabled the tadpole to become a powerful in vivo model forinvestigating, at a high resolution, how environmental chemicalscan affect developing neurons. For instance, a study using the Ranapipiens tadpole has shown that chronically exposing tectal neuronsto low, sub-micromolar levels of lead decreases both RGC axonarbor area and branchtip number. The same group showed thatacute lead exposure weakens synaptic transmission between RGCaxons and tectal dendrites (Cline et al., 1996a). More recently, theXenopus tadpole was used to characterize the effects of sub-lethalconcentrations of methylisothiazolinone (MIT; a biocide commonlyused in several cosmetics, including shampoo) on many aspects ofnervous system function (Spawn and Aizenman, 2012). Forexample, overall visual system function was tested using protocolsdesigned to characterize tectum-dependent and thalamus-dependent visual behaviors (Dong et al., 2009). MIT-exposedtadpoles displayed deficits in only the tectum-dependent visualbehavior, suggesting a malfunction in retinotectal synaptictransmission. Although no differences were observed in synaptictransmission between RGC inputs and tectal neurons in the MIT-treated tadpoles, the pattern of the recurrent tecto-tectalconnectivity – which is activated by RGC inputs – (Fig. 1C) wasaltered in a way that suggests lack of circuit refinement. At thesingle-neuron level, no differences in intrinsic excitability andsynaptic strengths were observed in MIT-treated tadpoles (Fig. 1D).In summary, the deficits in tectum-dependent visual behavior andunrefined tecto-tectal connectivity, combined with the absence ofnoticeable changes in intrinsic or synaptic properties, suggest thatchronic MIT exposure causes problems at the circuit level and notat the single-cell level. For neurotoxicology research, this studyexemplifies how chronic exposure to concentrations of a chemicalwith no noticeable effects on either survival or morphology canstill compromise a developing neural circuit. Overall, this study

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demonstrates the level of detail at which neurons and neural circuitscan be assayed using the Xenopus tadpole, and, more specifically,how a deficit in a behavior can be tracked down and studied at thecircuit and single-neuron level.

A tadpole model for epileptic seizureIn addition to environmental toxins, developing circuits areparticularly susceptible to epileptic activity. A protocol to reliablyinduce controlled seizures in the Xenopus tadpole has beendeveloped (Hewapathirane et al., 2008) and has already led to afundamental insight into how endogenous polyamines can play aneuroprotective role in response to an epileptic seizure (Bell et al.,2011).

The development of the tadpole model for studying epilepticseizures began with a detailed characterization of the ability ofseveral different classes of chemoconvulsants to reliably induceseizures in stage-47 tadpoles. Several different classes of knownconvulsants were tested: GABA-receptor antagonists[pentylenetetrazole (PTZ), picrotoxin and bicuculline], glutamatereceptor agonists (kainate), muscarinic receptor agonists(pilocarpine) and potassium channel inhibitors (4-aminopyridine)(Hewapathirane et al., 2008). All of these convulsants seemed toproduce a common type of behavioral seizure in tadpoles. Thebehavior commences with intermittent bouts of rapid swimming,followed by immobility, deviations from the normal head-down tail-up posture, and lateral movements of the head, followed ultimatelyby full-blown seizure behavior – C-shaped contractions evoked byabnormal unilateral axial muscle contractions that are so strongthat they result in the entire tadpole displaying a stereotypical ‘C’shape. Because all of the different classes of convulsant inducedthe same type of seizure, it was concluded that this is a ‘true’ seizurerather than the effects of a particular drug on motor function. TheGABA receptor antagonist PTZ was determined to be the optimalchemoconvulsant for the tadpole seizure model because it reliablyinduces the stereotypical C-shape contractions at doses that areneither lethal nor toxic, and in vivo field potential recordings inthe optic tectum revealed robust epileptiform activity, i.e. high-amplitude spiking, in response to PTZ application. Thisepileptiform activity can be blocked completely by administrationof the anti-epileptic drug valproate. An advantage of this model isthat immobilization of the tadpole for electrophysiology or imagingexperiments can be achieved using reversible paralytics or agarimmersion, thereby allowing seizures to be studied in the absenceof anesthetic agents (Hewapathirane et al., 2008).

In a recent study by Bell et al. (Bell et al., 2011) involving aprotocol consisting of two consecutive PTZ-induced seizures anda combination of behavioral, electrophysiological andpharmacological experiments, it was shown that the first initialPTZ-induced seizure in a tadpole increases the production ofpolyamines [an observation that had also been reported in a rodentseizure model (Hayashi et al., 1993)]. Elevated polyamine levels werefound to boost the production and release of the inhibitorytransmitter GABA, which rendered tadpoles less prone to futureseizures. Similarly, exposing tadpoles to enhanced visual stimulationled to increased GABA levels in the tectum, providing anothercompelling example of how GABA can function in a homeostaticmanner in response to abnormally high levels of circuit activity(Miraucourt et al., 2012).

Tadpole models for the study of autism spectrumdisordersAutism spectrum disorders (ASD) are paradoxical: the syndromeswithin this group present with a highly recognizable set ofsymptoms, yet they can be caused by a diverse array of geneticabnormalities and environmental insults, such as prenatal infection,hormonal exposure and teratogens (Newschaffer et al., 2007;Abrahams and Geschwind, 2008). Mutations in more than 40 geneshave been shown to increase susceptibility to ASD, yet none of thesemutations are completely penetrant, i.e. cause ASD with 100%probability (Lichtenstein et al., 2010; Neale et al., 2012).Furthermore, although the defining symptoms of ASD, such asdeficits in language, social interactions and personal interests aremanifested at the highest cognitive levels, the etiology of ASD hasbeen linked to abnormalities in surprisingly fundamental aspectsof nervous system functioning and development. This includesdefects in synaptic plasticity (Krey and Dolmetsch, 2007; Markramet al., 2008; Bhakar et al., 2012), inhibition/excitation balance (Perryet al., 2007; Markram and Markram, 2010; Marín, 2012),microcircuitry organization (Geschwind, 2009) and neuron-gliainteractions (Abrahams and Geschwind, 2008), as well as long-range underconnectivity and local overconnectivity, as aconsequence of altered axon guidance and dendritic arborization(Rinaldi et al., 2008; Geschwind, 2009). These features suggest thatASD represents a uniquely human response to a broad class ofdevelopmental dysregulations (Peça and Feng, 2012) and, therefore,the mechanisms of ASD are likely to be successfully addressed inanimal models not necessarily capable of expressing mostbehavioral and cognitive symptoms of ASD (Patterson, 2011). Theseanimal models would include mammals, but also fish (Tropepe andSive, 2003; Kabashi et al., 2011), birds (Panaitof, 2012), insects(Gatto and Broadie, 2011) and amphibians.

With this in mind, a successful experimental approach inXenopus would entail looking directly at the changes caused byknown ASD-associated developmental perturbations at the cellularand network levels. One of the unique benefits of the tadpole isthe ease at which gene expression can be altered in individualneurons, and the convenience of registering the consequences ofthese perturbations in vivo, allowing a way to differentiate betweencell-autonomous and network-level effects of ASD-associatedmutations. As a good example, when a wild-type human MeCP2gene [a mutation in this gene causes Rett syndrome in humans,and is strongly comorbid with ASD (Samaco and Neul, 2011)] wasoverexpressed in Xenopus tectal neurons in vivo, these neuronswere found to develop fewer, albeit longer, dendrites comparedwith normal tectal cells (Marshak et al., 2012). Hence, in Xenopus,as in humans and rodents, variations in MeCP2 activity causeredistribution between close- and long-range networkconnections, which is one of the landmark circuit abnormalitiesin ASD (Geschwind, 2009). This work also illustrates that keytranscription regulators are sufficiently conserved betweenXenopus and humans (Amir et al., 1999), allowing the humanMeCP2 gene to interact (Marshak et al., 2012) with native Xenopuspathways (Stancheva et al., 2003).

Although not every gene linked to ASD in humans (Abrahamsand Geschwind, 2008; Neale et al., 2012) has been identified andstudied in Xenopus thus far (Hellsten et al., 2010), a large proportionhave been; see Table 1 for a list. Among all ASD-related genetic

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conditions, the one that is most researched in a Xenopus model isFragile X syndrome, which is linked to the deactivation of a singlegene, FMR1 (Abrahams and Geschwind, 2008; Levenga et al., 2010;Bhakar et al., 2012). The tadpole can be easily employed for studyingassociated network pathology via single-gene manipulation in thedeveloping embryo. It has already been shown that manipulationof the fmr1 gene in the developing Xenopus embryo disrupts propersomite formation (Huot et al., 2012), and it will be interesting toobserve how altered FMR1 expression affects developing neuralcircuits in humans. The Fragile-X-related genes are well describedin Xenopus (both X. tropicalis and X. laevis), with frogs having fewerhomologs within the gene family (fmrp and fxr1p) (Lim et al., 2005)than do humans (FMRP, FXR1P and FXR2P), as well as fewerisoforms per gene (Huot et al., 2005). Even so, it has beendemonstrated that antibodies to human FMRP and FXRP proteinsare effective against Xenopus proteins (Blonden et al., 2005; Huotet al., 2005), and that expression of human FMRP or FXRP rescuesfmrp and fxr1p knockdown phenotypes in Xenopus, respectively(Huot et al., 2005; Gessert et al., 2010).

Neuroligins and neurexins (synaptic adhesion proteins), togetherwith Shank and PSD-95 scaffolding proteins, make up anotherfunctional group of proteins that share high degrees of homologybetween Xenopus and mammals (Ichtchenko et al., 1996; Chen etal., 2010; Gessert et al., 2011), have mutations in correspondinggenes linked to ASD in human patients (Jamain et al., 2003; Fenget al., 2006; Marín, 2012), and have been successfully studied inXenopus. When neuroligin-1 function was disrupted in Xenopus

tectal neurons by either overexpression of mutant forms of mouseNlg-1, competitive saturation of native Nlg-1 extracellular domainswith soluble neurexin motifs, or a knock-down of native Nlg-1 withmorpholinos, the dendritic tree maturation profile for theseneurons was altered. Specifically, the tectal neurons failed toestablish new synapses with RGC axons, demonstrated highermotility of dendritic filopodia, and had a simplified and immaturedendritic arbor morphology overall (Chen et al., 2010). Moreover,by comparing effects of these perturbations on the dynamics ofdendritic arbor development, the authors managed to convincinglyreconstruct the pattern of protein interactions occurring duringfilopodia stabilization and synapse formation. This work providesanother example of how in situ time-lapse imaging in a live tadpolebrain (Bestman et al., 2012; Hossain et al., 2012) can be combinedwith genetic manipulations (Bestman et al., 2006; Bestman andCline, 2008; Liu and Haas, 2011) and electrophysiology (Pratt andAizenman, 2007) to dissect the functional role of target synapticproteins and their influence on network formation (Chen and Haas,2011).

Finally, Xenopus modeling can help us to probe the link betweenASD and immune activation in the brain (Deverman and Patterson,2009). Although increases in glia activation and levels of pro-inflammatory cytokines have been observed in individuals withASD, it is unclear whether these phenomena contribute to thecause, or are a consequence of the disorder (Cohly and Panja, 2005).When tadpoles were chronically exposed to interleukins (IL-1β, IL-6) or tumor necrosis factor (TNFα), tectal neurons were

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Table 1. Genes that are linked to ASD in humans, and that have been identified and studied in Xenopus

Human gene Gene function Association with ASD Studies in Xenopus MeCP2 Transcription modulator Linked to Rett syndrome (Samaco and

Neul, 2011) Expression (Stancheva et al., 2003); effects on cell

morphology (Marshak et al., 2012). See the text

for details.

FMR1 (aka

FMRP), FXR1,

FXR2

Regulator of translation and mRNA

shuttling Linked to Fragile X syndrome (Spencer et

al., 2006; Abrahams and Geschwind,

2008; Levenga et al., 2010; Guo et al., 2011; Bhakar et al., 2012)

Genetics (Lim et al., 2005; Huot et al., 2012);

expression (Blonden et al., 2005); effects on

development (Huot et al., 2005; Gessert et al., 2010; Huot et al., 2012). See the text for details.

NLGN Neuroligins (synaptic adhesion proteins) (Jamain et al., 2003) Effects on cell morphology development (Chen et

al., 2010). See the text for details.

NRX Neurexins (synaptic adhesion proteins) (Feng et al., 2006) Effects on cell morphology development (Chen et

al., 2010). See the text for details.

MEF2 Transcription factor Linked to Fragile X syndrome (Tsai et al.,

2012) Effects on cell functional and morphological

maturation (Chen et al., 2012)

Shank Synaptic scaffolding proteins (Abrahams and Geschwind, 2008; Peça and Feng, 2012)

Expression pattern (Gessert et al., 2011)

WNT-2 Signaling protein, developmental

regulator (Wassink et al., 2001) Expression pattern, effects on development

(Landesman and Sokol, 1997)

CACNA1C CaV1.2 channel gene (voltage-dependent

ion channel) Linked to Timothy syndrome (Krey and

Dolmetsch, 2007) Expression pattern (Lewis et al., 2009)

IR Insulin receptor (Chiu and Cline, 2010; Stern, 2011) Effects on cell function and morphology (Chiu et

al., 2008)

PER1 Circadian gene (member of a Period family)

(Nicholas et al., 2007) Expression regulation (Zhuang et al., 2000)

GRIK2 Kainate glutamate receptor (ionotropic

receptor, aka GluR6, GluK2) (Jamain et al., 2002) Channel properties (Ishimaru et al., 1996)

PTEN Cell cycle regulator, tumor suppressor

gene Linked to Cowden syndrome (Butler et al.,

2005) Effects on development (Ueno et al., 2006)

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overconnected (based on the electrophysiological evidence),synapses seemed more mature (based on the AMPA:NMDA ratio),and animals had abnormal sensory processing, and were susceptibleto seizures (Lee et al., 2010), confirming that immune activationalone can trigger some components of the ASD phenotype.

ConclusionsOverall, these experiments underscore the power of the tadpolemodel in addressing a question from the molecular to the systemslevel (Fig.  1). As discussed in the Introduction, there are manyfactors that make the Xenopus laevis tadpole a particularly well-suited model for studying neurodevelopmental disorders. The onlymajor weakness of the X. laevis model is that, because this speciesis tetraploid, meaning that they carry four copies of each gene,genetic manipulations are relatively difficult compared withmanipulations of diploid genomes. Recent advances in transgenictechniques have made it possible to generate transgenic X. laevistadpoles (Kroll and Amaya, 1996). These transgenic tadpoles havebeen used with much success (Kroll and Amaya, 1996; Marshak etal., 2007; Takagi et al., 2013), a relevant example being thegeneration of tadpoles that expressed dominant-negative TrkB(BDNF/neurotrophin receptor) exclusively in retinal ganglion cells(Marshak et al., 2007). Still, there are issues that remain: with fourcopies of any given gene, it is virtually impossible to completelyknock out its expression. Instead, researchers have resorted toexpressing dominant-negative mutations (Hawley et al., 1995;Coen et al., 2007) that effectively inhibit the endogenous functionof a gene, but are often non-specific, inhibiting more than a singlegene product. In addition to the expression of dominant-negativemutations, RNA interference (RNAi) (Miskevich et al., 2006) andmorpholinos (Root et al., 2008; Sharma and Cline, 2010) have beenused successfully in Xenopus to knock down gene expression. Whatcould prove to be an even better vertebrate model, especially forgenetics, is the only diploid species of Xenopus, Xenopus tropicalis(Amaya et al., 1998). Being diploid renders the X. tropicalis moreamenable to genetic manipulations. Other than the difference inthe number of copies of genes, these two species are quite similar.Thus, as a vertebrate model, X. tropicalis offers all the advantagesof the X. laevis model without the experimental complications thatare inherent to a polyploid genome. Hence, the X. tropicalis tadpolehas been predicted to be the animal model of the future (Amayaet al., 1998).

In conclusion, the Xenopus tadpole has been and continues tobe a powerful and prolific model to study, at many levels, bothnormal and abnormal neural development. The ability to studydeveloping neural circuits at these different levels and with highresolution allows the tadpole to contribute greatly to theidentification of currently unknown targets to treatneurodevelopmental disorders in humans.ACKNOWLEDGEMENTSWe thank Carlos Aizenman, Harold Bergman, Jane Sullivan, Lulu Tsai and RyanMaloney for helpful comments and suggestions.

COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

FUNDINGK.G.P. is supported by a grant from the National Institute of General MedicalSciences (P30 GM103398) from the National Institutes of Health. A.S.K. is fundedby NIH R01 EY019578-03 grant (PI: Carlos Aizenman).

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