New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome.

OBJECTIVE: To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT2 receptor agonist fenfluramine. METHODS: We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled γ-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae. RESULTS: Homozygous mutant (scn1labmut/mut ) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean = 7.5 per 20-minute recording; P < .0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1labmut/mut larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P < .001; n = 15 mutant, n = 16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1labmut/mut larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1labmut/mut larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1labmut/mut larval brains that were rescued by fenfluramine but not diazepam. SIGNIFICANCE: Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1labmut/mut brain, and present first-time evidence for potential disease modification by fenfluramine.

Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish.

BACKGROUND: Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined. RESULTS: We aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon. CONCLUSIONS: Herein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus-unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates.

Astroglial DJ-1 over-expression up-regulates proteins involved in redox regulation and is neuroprotective in vivo.

DJ-1, a Parkinson's disease-associated protein, is strongly up-regulated in reactive astrocytes in Parkinson's disease. This is proposed to represent a neuronal protective response, although the mechanism has not yet been identified. We have generated a transgenic zebrafish line with increased astroglial DJ-1 expression driven by regulatory elements from the zebrafish GFAP gene. Larvae from this transgenic line are protected from oxidative stress-induced injuries as caused by MPP+, a mitochondrial complex I inhibitor shown to induce dopaminergic cells death. In a global label-free proteomics analysis of wild type and transgenic larvae exposed to MPP+, 3418 proteins were identified, in which 366 proteins were differentially regulated. In particular, we identified enzymes belonging to primary metabolism to be among proteins affected by MPP+ in wild type animals, but not affected in the transgenic line. Moreover, by performing protein profiling on isolated astrocytes we showed that an increase in astrocytic DJ-1 expression up-regulated a large group of proteins associated with redox regulation, inflammation and mitochondrial respiration. The majority of these proteins have also been shown to be regulated by Nrf2. These findings provide a mechanistic insight into the protective role of astroglial up-regulation of DJ-1 and show that our transgenic zebrafish line with astrocytic DJ-1 over-expression can serve as a useful animal model to understand astrocyte-regulated neuroprotection associated with oxidative stress-related neurodegenerative disease.

Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses.

Inhibitory glycine receptors (GlyRs) are pentameric ligand-gated anion channels with major roles in startle disease/hyperekplexia (GlyR α1), cortical neuronal migration/autism spectrum disorder (GlyR α2), and inflammatory pain sensitization/rhythmic breathing (GlyR α3). However, the role of the GlyR α4 subunit has remained enigmatic, because the corresponding human gene (GLRA4) is thought to be a pseudogene due to an in-frame stop codon at position 390 within the fourth membrane-spanning domain (M4). Despite this, a recent genetic study has implicated GLRA4 in intellectual disability, behavioral problems and craniofacial anomalies. Analyzing data from sequenced genomes, we found that GlyR α4 subunit genes are predicted to be intact and functional in the majority of vertebrate species-with the exception of humans. Cloning of human GlyR α4 cDNAs excluded alternative splicing and RNA editing as mechanisms for restoring a full-length GlyR α4 subunit. Moreover, artificial restoration of the missing conserved arginine (R390) in the human cDNA was not sufficient to restore GlyR α4 function. Further bioinformatic and mutagenesis analysis revealed an additional damaging substitution at K59 that ablates human GlyR α4 function, which is not present in other vertebrate GlyR α4 sequences. The substitutions K59 and X390 were also present in the genome of an ancient Denisovan individual, indicating that GLRA4 has been a pseudogene for at least 30,000-50,000 years. In artificial synapses, we found that both mouse and gorilla α4ß GlyRs mediate synaptic currents with unusually slow decay kinetics. Lastly, to gain insights into the biological role of GlyR α4 function, we studied the duplicated genes glra4a and glra4b in zebrafish. While glra4b expression is restricted to the retina, using a novel tol2-GAL4FF gene trap line (SAIGFF16B), we found that the zebrafish GlyR α4a subunit gene (glra4a) is strongly expressed in spinal cord and hindbrain commissural neurones. Using gene knockdown and a dominant-negative GlyR α4aR278Q mutant, we found that GlyR α4a contributes to touch-evoked escape behaviors in zebrafish. Thus, although GlyR α4 is unlikely to be involved in human startle responses or disease states, this subtype may contribute to escape behaviors in other organisms.

Optimization of a Neurotoxin to Investigate the Contribution of Excitatory Interneurons to Speed Modulation In Vivo.

Precise control of speed during locomotion is essential for adaptation of behavior in different environmental contexts [1-4]. A central question in locomotion lies in understanding which neural populations set locomotor frequency during slow and fast regimes. Tackling this question in vivo requires additional non-invasive tools to silence large populations of neurons during active locomotion. Here we generated a stable transgenic line encoding a zebrafish-optimized botulinum neurotoxin light chain fused to GFP (BoTxBLC-GFP) to silence synaptic output over large populations of motor neurons or interneurons while monitoring active locomotion. By combining calcium imaging, electrophysiology, optogenetics, and behavior, we show that expression of BoTxBLC-GFP abolished synaptic release while maintaining characterized activity patterns and without triggering off-target effects. As chx10(+) V2a interneurons (V2as) are well characterized as the main population driving the frequency-dependent recruitment of motor neurons during fictive locomotion [5-14], we validated our silencing method by testing the effect of silencing chx10(+) V2as during active and fictive locomotion. Silencing of V2as selectively abolished fast locomotor frequencies during escape responses. In addition, spontaneous slow locomotion occurred less often and at frequencies lower than in controls. Overall, this silencing approach confirms that V2a excitation is critical for the production of fast stimulus-evoked swimming and also reveals a role for V2a excitation in the production of slower spontaneous locomotor behavior. Altogether, these results establish BoTxBLC-GFP as an ideal tool for in vivo silencing for probing the development and function of neural circuits from the synaptic to the behavioral level.

BAC transgenic zebrafish reveal hypothalamic enhancer activity around obesity associated SNP rs9939609 within the human FTO gene.

Single Nucleotide Polymorphisms in FTO intron 1 have been associated with obesity risk, leading to the hypothesis that FTO is the obesity-related gene. However, other studies have shown that the FTO gene is part of the regulatory domain of the neighboring IRX3 gene and that enhancers in FTO intron 1 regulate IRX3. While Irx3 activity was shown to be necessary in the hypothalamus for the metabolic function of Irx3 in mouse, no enhancers with hypothalamic activity have been demonstrated in the risk-associated region within FTO. In order to identify potential enhancers at the human FTO locus in vivo, we tested regulatory activity in FTO intron 1 using BAC transgenesis in zebrafish. A minimal gata2 promoter-GFP cassette was inserted 1.3 kb upstream of the obesity associated SNP rs9939609 in a human FTO BAC plasmid. In addition to the previously identified expression domains in notochord and kidney, human FTO BAC:GFP transgenic zebrafish larvae expressed GFP in the ventral posterior tuberculum, the posterior hypothalamus and the anterior brainstem, which are also expression domains of zebrafish irx3a. In contrast, an in-frame insertion of a GFP cassette at the FTO start codon resulted in weak ubiquitous GFP expression indicating that the promoter of FTO does likely not react to enhancers located in the obesity risk-associated region.

Deletion of a kinesin I motor unmasks a mechanism of homeostatic branching control by neurotrophin-3.

Development and function of highly polarized cells such as neurons depend on microtubule-associated intracellular transport, but little is known about contributions of specific molecular motors to the establishment of synaptic connections. In this study, we investigated the function of the Kinesin I heavy chain Kif5aa during retinotectal circuit formation in zebrafish. Targeted disruption of Kif5aa does not affect retinal ganglion cell differentiation, and retinal axons reach their topographically correct targets in the tectum, albeit with a delay. In vivo dynamic imaging showed that anterograde transport of mitochondria is impaired, as is synaptic transmission. Strikingly, disruption of presynaptic activity elicits upregulation of Neurotrophin-3 (Ntf3) in postsynaptic tectal cells. This in turn promotes exuberant branching of retinal axons by signaling through the TrkC receptor (Ntrk3). Thus, our study has uncovered an activity-dependent, retrograde signaling pathway that homeostatically controls axonal branching.

Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena.

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3(Y) transgenic fish, and Sox3(Y) loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.

Different combinations of Notch ligands and receptors regulate V2 interneuron progenitor proliferation and V2a/V2b cell fate determination.

The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand-receptor combinations that occur concurrently during development in zebrafish.

Transgenic tools to characterize neuronal properties of discrete populations of zebrafish neurons.

The developing nervous system consists of a variety of cell types. Transgenic animals expressing reporter genes in specific classes of neuronal cells are powerful tools for the study of neuronal network formation. We generated a wide variety of transgenic zebrafish that expressed reporter genes in specific classes of neurons or neuronal progenitors. These include lines in which neurons of specific neurotransmitter phenotypes expressed fluorescent proteins or Gal4, and lines in which specific subsets of the dorsal progenitor domain in the spinal cord expressed fluorescent proteins. Using these, we examined domain organization in the developing dorsal spinal cord, and found that there are six progenitor domains in zebrafish, which is similar to the domain organization in mice. We also systematically characterized neurotransmitter properties of the neurons that are produced from each domain. Given that reporter gene expressions occurs in a wide area of the nervous system in the lines generated, these transgenic fish should serve as powerful tools for the investigation of not only the neurons in the dorsal spinal cord but also neuronal structures and functions in many other regions of the nervous system.

Alleviation of seipinopathy-related ER stress by triglyceride storage.

Mutations affecting the N-glycosylation site in Berardinelli-Seip lipodystrophy (BSCL)-associated gene BSCL2/seipin lead to a dominantly inherited spastic paraplegia termed seipinopathy. While the loss of function of seipin leads to severe congenital lipodystrophy, the effects of seipin N-glycosylation mutations on lipid balance in the nervous system are unknown. In this study, we show that expression of seipin N-glycosylation mutant N88S led to decreased triglyceride (TG) content in astrocytoma and motor neuron cell lines. This was corrected by supplementation with exogenous oleic acid. Upon oleic acid loading, seipin N88S protein was relocated from the endoplasmic reticulum (ER) to the surface of lipid droplets and this was paralleled by alleviation of ER stress induced by the mutant protein. This effect was not limited to seipin N88S, as oleic acid loading also reduced tunicamycin-induced ER stress in motor neuron cells. Furthermore, both seipin N88S and tunicamycin-induced ER stress were decreased by inhibiting lipolysis, suggesting that lipid droplets protected neuronal cells from ER stress. In developing zebrafish larvae, seipin N88S expression led to TG imbalance and reduced spontaneous free swimming. Importantly, supplementation with exogenous oleic acid reduced ER stress in the zebrafish head and increased fish motility. We propose that the decreased TG content contributes to the pathology induced by seipin N88S, and that rescuing TG levels may provide a novel therapeutic strategy in seipinopathy.

The medaka zic1/zic4 mutant provides molecular insights into teleost caudal fin evolution.

Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.

Transposon-mediated BAC transgenesis in zebrafish.

Bacterial artificial chromosomes (BACs) are widely used in studies of vertebrate gene regulation and function because they often closely recapitulate the expression patterns of endogenous genes. Here we report a step-by-step protocol for efficient BAC transgenesis in zebrafish using the medaka Tol2 transposon. Using recombineering in Escherichia coli, we introduce the iTol2 cassette in the BAC plasmid backbone, which contains the inverted minimal cis-sequences required for Tol2 transposition, and a reporter gene to replace a target locus in the BAC. Microinjection of the Tol2-BAC and a codon-optimized transposase mRNA into fertilized eggs results in clean integrations in the genome and transmission to the germline at a rate of ∼15%. A single person can prepare a dozen constructs within 3 weeks, and obtain transgenic fish within approximately 3-4 months. Our protocol drastically reduces the labor involved in BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates.

Transposon-mediated BAC transgenesis in zebrafish and mice.

BACKGROUND: Bacterial artificial chromosomes (BACs) are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications. RESULTS: We took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a approximately 70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations. CONCLUSION: The Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.

Transgenesis in zebrafish with the tol2 transposon system.

The zebrafish (Danio rerio) is a useful model for genetic studies of vertebrate development. Its embryos are transparent and develop rapidly outside the mother, making it feasible to visualize and manipulate specific cell types in the living animal. Zebrafish is well suited for transgenic manipulation since it is relatively easy to collect large numbers of embryos from adult fish. Several approaches have been developed for introducing transgenes into the zebrafish germline, from the injection of naked DNA to transposon-mediated integration. In particular, the Tol2 transposable element has been shown to create insertions in the zebrafish genome very efficiently. By using Tol2, gene trap and enhancer trap vectors containing the GFP reporter gene or yeast transcription activator Gal4 gene have been developed. Here we outline methodology for creating transgenic zebrafish using Tol2 vectors, and their applications to visualization and manipulation of specific tissues or cells in vivo and for functional studies of vertebrate neural circuits.

A novel conserved evx1 enhancer links spinal interneuron morphology and cis-regulation from fish to mammals.

Spinal interneurons are key components of locomotor circuits, driving such diverse behaviors as swimming in fish and walking in mammals. Recent work has linked the expression of evolutionarily conserved transcription factors to key features of interneurons in diverse species, raising the possibility that these interneurons are functionally related. Consequently, the determinants of interneuron subtypes are predicted to share conserved cis-regulation in vertebrates with very different spinal cords. Here, we establish a link between cis-regulation and morphology of spinal interneurons that express the Evx1 homeodomain transcription factor from fish to mammals. Using comparative genomics, and complementary transgenic approaches, we have identified a novel enhancer of evx1, that includes two non-coding elements conserved in vertebrates. We show that pufferfish evx1 transgenes containing this enhancer direct reporter expression to a subset of spinal commissural interneurons in zebrafish embryos. Pufferfish, zebrafish and mouse evx1 downstream genomic enhancers label selectively Evx1(+) V0 commissural interneurons in chick and rat embryos. By dissecting the zebrafish evx1 enhancer, we identify a role for a 25 bp conserved cis-element in V0-specific gene expression. Our findings support the notion that spinal interneurons shared between distantly related vertebrates, have been maintained in part via the preservation of highly conserved cis-regulatory modules.

Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.

Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P element-mediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.

Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase.

This study links natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses.

Refining GAL4-driven transgene expression in Drosophila with a GAL80 enhancer-trap.

We constructed an enhancer-trap element, P[GAL80], that encodes the yeast GAL80 repressor to refine expression of transgenes driven by the binary GAL4/UAS system. GAL80 blocks GAL4 activity by binding to its transcriptional activation domain. We screened GAL80 enhancer-traps for repression of GAL4-induced green fluorescent protein (GFP) in the intact larval nervous system. We selected one line that repressed GFP in a large set of cholinergic neurons. This line was used to refine GFP expression from a set of over 200 neurons to a subset of 20 neurons in a preselected GAL4 line. Expression of tetanus neurotoxin, a potent blocker of neurotransmitter release, in these 20 neurons reproduced an aberrant larval turning behavior previously assigned to the parental set of 200 neurons. Our results suggest that targeted GAL80 expression could become a useful means of spatially refining transgene expression in Drosophila.

Targeted expression of tetanus toxin reveals sets of neurons involved in larval locomotion in Drosophila.

The Drosophila larva is widely used for studies of neuronal development and function, yet little is known about the neuronal basis of locomotion in this model organism. Drosophila larvae crawl over a plain substrate by performing repetitive waves of forward peristalsis alternated by brief episodes of head swinging and turning. To identify sets of central and peripheral neurons required for the spatial or temporal pattern of larval locomotion, we blocked neurotransmitter release from defined populations of neurons by targeted expression of tetanus toxin light chain (TeTxLC) with the GAL4/UAS system. One hundred fifty GAL4 lines were crossed to a UAS-TeTxLC strain and a motion-analysis system was used to identify larvae with abnormal movement patterns. Five lines were selected that show discrete locomotor defects (i.e., increased turning and pausing) and these defects are correlated with diverse sets of central neurons. One line, 4C-GAL4, caused an unusual circling behavior that is correlated with approximately 200 neurons, including dopaminergic and peptidergic interneurons. Expression of TeTxLC in all dopaminergic and serotonergic but not in peptidergic neurons, caused turning deficits that are similar to those of 4C-GAL4/TeTxLC larvae. The results presented here provide a basis for future genetic studies of motor control in the Drosophila larva.