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It is with great pleasure that we introduce this special issue of the Journal of Neurogenetics honouring Professor Marla Sokolowski’s career and outstanding scientific achievements. Marla’s foundat...

Drosophilacourtship studies have elucidated several principles of the neurogenetic organization of complex behavior. Through an integration across sensory modalities, males perform stereotypic patterns of chasing, courtship song production, and copulation attempts. Here we report a serendipitous finding that intense light not only enhances courtship toward female targets but also triggers unexpected courtship behaviors among male flies. Strikingly, in wild-type male-only chambers, we observed extreme behavioral manifestations, such as “chaining” and “wheeling”, resembling previously reported male-male courtship behaviors infruitlessmutants and in transformants with ectopicmini-white+overexpression. This male-male courtship was greatly diminished in a variety of visual system mutants, including disrupted phototransduction (norpA), eliminated eye-color screening pigments (white), or deletion of the R7 photoreceptor cells (sevenless). However, light-induced courtship was unhampered in wing-cut flies, despite their inability to produce courtship song, a major acoustic signal during courtship. Unexpectedly the olfactory mutantsorcoandsbldisplayed unrestrained male-male courtship. Particularly,orcomales attained maximum courtship scores under either dim or intense light conditions. Together, our observations support the notion that the innate male courtship behavior is restrained by olfactory cues under normal conditions but can be unleashed by strong visual stimulation inDrosophila.

Pyridox (am) ine 5'-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting dietary vitamin B6 (VB6) to pyridoxal 5'-phosphate (PLP), the biologically active form of VB6 and involved in the synthesis of neurotransmitters including γ-aminobutyric acid (GABA), dopamine, and serotonin. In humans, PNPO mutations have been increasingly identified in neonatal epileptic encephalopathy and more recently also in early-onset epilepsy. Till now, little is known about the neurobiological mechanisms underlying PNPO-deficiency-induced seizures due to the lack of animal models. Previously, we identified a c.95 CA missense mutation in sugarlethal (sgll)-the Drosophila homolog of human PNPO (hPNPO)-and found mutant (sgll95) flies exhibiting a lethal phenotype on a diet devoid of VB6. Here, we report the establishment of both sgll95 and ubiquitous sgll knockdown (KD) flies as valid animal models of PNPO-deficiency-induced epilepsy. Both sgll95 and sgll KD flies exhibit spontaneous seizures before they die. Electrophysiological recordings reveal that seizures caused by PNPO deficiency have characteristics similar to that in flies treated with the GABA antagonist picrotoxin. Both seizures and lethality are associated with low PLP levels and can be rescued by ubiquitous expression of wild-type sgll or hPNPO, suggesting the functional conservation of the PNPO enzyme between humans and flies. Results from cell type-specific sgll KD further demonstrate that PNPO in the brain is necessary for seizure prevention and survival. Our establishment of the first animal model of PNPO deficiency will lead to better understanding of VB6 biology, the PNPO gene and its mutations discovered in patients, and can be a cost-effective system to test therapeutic strategies.

Significance Both genetic and environmental factors contribute to epilepsy. Understanding their contributions and interactions helps disease management. However, it is often challenging to study gene–environment interaction in humans due to their heterogeneous genetic background and less controllable environmental factors. The fruit fly, Drosophila melanogaster , has been proven to be a powerful model to study human diseases, including epilepsy. We generated knock-in flies carrying different epilepsy-associated pyridox(am)ine 5 ′ -phosphate oxidase ( PNPO ) alleles and studied the developmental, behavioral, electrophysiological, and fitness effects of each mutant allele under different dietary conditions. We showed that phenotypes in knock-in flies are allele and diet dependent, providing clues for timely and specific diet interventions. Our results offer biological insights into mechanisms underlying phenotypic variations and specific therapeutic strategies.

Previous studies have demonstrated that mutations of the Drosophila planar cell polarity gene prickle (pk) result in altered microtubule-mediated vesicular transport in larval motor axons, as well as adult neuronal circuit hyperexcitability and epileptic behavior. It is also known that mutant alleles of the prickle-prickle (pkpk) and prickle-spiny-legs (pksple) isoforms differ in phenotype but display isoform counterbalancing effects in heteroallelic pkpk/pksple flies to ameliorate adult motor circuit and behavioral hyperexcitability. We have further investigated the larval neuromuscular junction (NMJ) and uncovered robust phenotypes in both pkpk and pksple alleles (heretofore referred to as pk and sple alleles, respectively), including synaptic terminal overgrowth, as well as irregular motor axon terminal excitability, poor vesicle release synchronicity, and altered efficacy of synaptic transmission. We observed significant increase in whole-cell excitatory junctional potential (EJP) in pk homozygotes, which was restored to near WT level in pk/sple heterozygotes. We further examined motor terminal excitability sustained by presynaptic Ca2+ channels, under the condition of pharmacological blockade of Na+ and K+ channel function. Such manipulation revealed extreme Ca2+ channel-dependent nerve terminal excitability in both pk and sple mutants. However, when combined in pk/sple heterozygotes, such terminal hyper-excitability was restored to nearly normal. Focal recording from individual synaptic boutons revealed asynchronous vesicle release in both pk and sple homozygotes, which nevertheless persisted in pk/sple heterozygotes without indications of isoform counter-balancing effects. Similarly, the overgrowth at NMJs was not compensated in pk/sple heterozygotes, exhibiting an extremity comparable to that in pk and sple homozygotes. Our observations uncovered differential roles of the pk and sple isoforms and their distinct interactions in the various structural and functional aspects of the larval NMJ and adult neural circuits.

The Drosophila mutant paraShu harbors a dominant, gain-of-function allele of the voltage-gated sodium channel gene, paralytic (para). The mutant flies display severe seizure-like phenotypes...

Hypersynchronous neural activity is a characteristic feature of seizures. Although many Drosophila mutants of epilepsy-related genes display clear behavioral spasms and motor unit hyperexcitability, field potential measurements of aberrant hypersynchronous activity across brain regions during seizures have yet to be described. Here, we report a straightforward method to observe local field potentials (LFPs) from the Drosophila brain to monitor ensemble neural activity during seizures in behaving tethered flies. High frequency stimulation across the brain reliably triggers a stereotypic sequence of electroconvulsive seizure (ECS) spike discharges readily detectable in the dorsal longitudinal muscle (DLM) and coupled with behavioral spasms. During seizure episodes, the LFP signal displayed characteristic large-amplitude oscillations with a stereotypic temporal correlation to DLM flight muscle spiking. ECS-related LFP events were clearly distinct from rest- and flight-associated LFP patterns. We further characterized the LFP activity during different types of seizures originating from genetic and pharmacological manipulations. In the ‘bang-sensitive’ sodium channel mutant bangsenseless (bss), the LFP pattern was prolonged, and the temporal correlation between LFP oscillations and DLM discharges was altered. Following administration of the pro-convulsant GABA(A) blocker picrotoxin, we uncovered a qualitatively different LFP activity pattern, which consisted of a slow (1-Hz), repetitive, waveform, closely coupled with DLM bursting and behavioral spasms. Our approach to record brain LFPs presents an initial framework for electrophysiological analysis of the complex brain-wide activity patterns in the large collection of Drosophila excitability mutants.

Pyridox(am)ine 5’-phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5’-phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters GABA and monoamines. Pathogenic variants in PNPO have been repeatedly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities, including developmental impairment and intellectual disability. It is unclear how seizure types and associated comorbidities are linked to specific PNPO alleles and to what degree diet can modify their expression. Furthermore, the molecular characteristics of PNPO variants have not been examined in model systems. Using CRISPR/Cas9, we generated four knock-in Drosophila alleles, hWT, hR116Q, hD33V, and hR95H, in which the endogenous Drosophila PNPO (sugarlethal) was replaced by wild-type human PNPO cDNA and epilepsy-associated variants corresponding to R116Q, D33V, and R95H, respectively. We examined these knock-in flies at the molecular, circuitry, and behavioral levels. Collectively, we found a wide range of phenotypes in an allele- and diet-dependent manner. Specifically, the D33V mutation reduces the mRNA level, R95H reduces the protein stability, and R116Q alters the protein localization of PNPO in the brain. D33V and R95H mutations lead to partial and complete lethality during development, respectively and R116Q and D33V mutations shorten lifespan. At the behavioral level, hD33V/hR95H trans-heterozygous flies are hypoactive on all tested diets whereas hR116Q flies show diet-dependent locomotor activities. At the circuitry level, hD33V homozygotes show rhythmic burst firing and hD33V/hR95H trans-heterozygotes exhibit spontaneous seizure discharges. In hR95H homozygotes rescued with PLP supplementation, we uncovered that PLP deficiency abolishes development and causes extreme seizures in adults. Lastly, genetic and electrophysiological analyses demonstrate that hWT/hR95H heterozygous flies are prone to seizures due to a dominant-negative effect of hR95H on hWT, highlighting the possibility that human R95H carriers may also be susceptible to epilepsy. Together, this study demonstrates that human PNPO variants interact with diet to contribute to phenotypic variations; and that the knock-in Drosophila model offers a powerful approach to systematically examine clinical manifestations and the underlying mechanisms of human PNPO deficiency.

Oxidative stress is thought to be a major contributor to aging processes. Here, we report differential effects on neurotransmission caused by loss-of-function mutations of Superoxide dismutase 1 (Sod1) and by paraquat (PQ) feeding in Drosophila. We demonstrated alterations in Sod1 mutants; the larval neuromuscular junction displayed supernumerary discharges and the adult giant-fiber escape pathway showed increased latency and poor response to repetitive high-frequency stimulation. Even though the concentrations used led to motor coordination defects and lethality, PQ feeding failed to reproduce such performance deficits in these larval and adult preparations, indicating mechanistic distinctions between these genetic and pharmacological manipulations of oxidative stress.

Hypersynchronous neural activity is a characteristic feature of seizures. Although many Drosophila mutants of epilepsy-related genes display clear behavioral spasms and motor unit hyperexcitability, field potential measurements of aberrant hypersynchronous activity across brain regions during seizures have yet to be described. Here, we report a straightforward method to observe local field potentials (LFPs) from the Drosophila brain to monitor ensemble neural activity during seizures in behaving tethered flies. High frequency stimulation across the brain reliably triggers a stereotypic sequence of electroconvulsive seizure (ECS) spike discharges readily detectable in the dorsal longitudinal muscle (DLM) and coupled with behavioral spasms. During seizure episodes, the LFP signal displayed characteristic large-amplitude oscillations with a stereotypic temporal correlation to DLM flight muscle spiking. ECS-related LFP events were clearly distinct from rest- and flight-associated LFP patterns. We further characterized the LFP activity during different types of seizures originating from genetic and pharmacological manipulations. In the ‘bang-sensitive’ sodium channel mutant bangsenseless (bss), the LFP pattern was prolonged, and the temporal correlation between LFP oscillations and DLM discharges was altered. Following administration of the pro-convulsant GABAA blocker picrotoxin, we uncovered a qualitatively different LFP activity pattern, which consisted of a slow (1-Hz), repetitive, waveform, closely coupled with DLM bursting and behavioral spasms. Our approach to record brain LFPs presents an initial framework for electrophysiological analysis of the complex brain-wide activity patterns in the large collection of Drosophila excitability mutants.

Oxidative stress is thought to be a major contributor to aging processes. Here, we report differential effects on neurotransmission caused by loss-of-function mutations of Superoxide dismutase (Sod) and by paraquat (PQ) feeding in Drosophila. We demonstrated alterations in Sod mutants; the larval neuromuscular junction displayed supernumerary discharges and the adult giant-fiber escape pathway showed increased latency and poor response to repetitive high-frequency stimulation. Even though the concentrations used led to motor coordination defects and lethality, PQ feeding failed to reproduce such performance deficits in these larval and adult preparations, indicating mechanistic distinctions between genetic and pharmacological manipulation of oxidative stress.

The reactive oxygen species (ROS)-scavenging enzyme Cu/Zn superoxide dismutase (SOD1) is an evolutionarily conserved mechanism for the maintenance of oxidative homeostasis, and missense mutations in the human SOD1 gene are associated with the motor neuron degenerative disease amyotrophic lateral sclerosis (ALS). Mutations in theDrosophila melanogasterSOD1 gene (Sod) shorten fly lifespan, attenuate motor function, and induce developmental mortality. We have previously found morphological and physiological defects at the neuromuscular junctions (NMJs) of hypomorphicSodn108and ALS modelSodG85Rmutant larvae. Here, we report genetic interactions causing striking modifications ofSodmutant phenotypes by mutations in the genePrickle(Pk), which are linked to planar cell polarity, epilepsy, and axonal transport disruptions.Pkis expressed as two isoformsprickle-spiny-legs(sple) andprickle-prickle(pk), which are each suppressed by specific hypomorphic mutations (sple1andpk1). Interestingly,Sodphenotypes are distinctly modified depending on both thePkisoform suppressed, and whether said suppression is heterozygous or homozygous. Heterozygoussple1andpk1improved the developmental survival ofSodmutants, whereas homozygoussple1andpk1drastically increased mortality. Further, only heterozygoussple1andpk1clearly ameliorated morphological defects at the neuromuscular junctions ofSodmutant larvae. Pharmacological treatment ofSodmutants reveals allele-specific motor neuron terminal hyperexcitability, characterized by synaptic transmissions of extended duration and abnormal presynaptic Ca2+transients. Heterozygoussple1mutation suppresses thisSodmutant terminal hyperexcitability, butpk1does not. We reversed this suppressive effect ofsple1by pharmacological blockade of Ca2+-activated K+channelslowpoke. Altogether, this study builds on our prior knowledge ofSodmutant development and physiology to show that axonal transport-linked gene mutations strikingly modifySodmutant phenotypes, providing strong evidence for a role of intracellular transport in alterations of neuromuscular morphology and physiology by SOD1 mutations.

We examined several sensory-motor processing circuits inDrosophilaacross the lifespan and uncovered distinctive age-resilient and age-vulnerable trajectories in their established functional properties. We observed relatively little deterioration toward the end of lifespan in the giant-fiber (GF) and downstream circuit elements responsible for the jump-and-flight escape reflex. In contrast, we found substantial age-dependent modifications in the performance of GF inputs and other circuits driving flight motoneuron activities. Importantly, in high temperature (HT)-reared flies (29 °C), the characteristic age-dependent progression of these properties was largely maintained, albeit over a compressed time scale, lending support for the common practice of expeditingDrosophilaaging studies by HT rearing. We discovered shortened lifespans inCu2+/Zn2+Superoxide Dismutase 1(Sod) mutant flies were accompanied by alterations distinct from HT-reared flies, highlighting differential effects of oxidative vs temperature stressors. This work also establishes several age-vulnerable parameters that may serve as quantitative neurophysiological landmarks for aging inDrosophila.

This special issue is dedicated to ‘Future Perspectives of Neurogenetics’ in honor of Troy D. Zars (1967–2018). It is intended to offer insight and inspiration for our understanding of how adaptive...

GCaMP imaging is widely employed for investigating neuronal Ca2+ dynamics. The Drosophila larval neuromuscular junction (NMJ) consists of three distinct types of motor terminals (type Ib, Is and II...

InDrosophila, molecular pathways affecting longevity have been extensively studied. However, corresponding neurophysiological changes underlying aging-related functional and behavioral deteriorations remain to be fully explored. We examined different motor circuits inDrosophilaacross the life span and uncovered distinctive age-resilient and age-vulnerable trajectories in their established functional properties. In the giant fiber (GF) and downstream circuit elements responsible for the jump-and-flight escape reflex, we observed relatively mild deterioration toward the end of the life span. In contrast, more substantial age-dependent modifications were seen in the plasticity of GF afferent processing, specifically in use dependence and habituation properties. In addition, there were profound changes in different afferent circuits that drive flight motoneuron activities, including flight pattern generation and seizure spike discharges evoked by electroconvulsive stimulation. Importantly, in high-temperature (HT)-reared flies (29°C), the general trends in these age-dependent trajectories were largely maintained, albeit over a compressed time scale, lending support for the common practice of HT rearing for expeditingDrosophilaaging studies. We discovered that shortened life spans inCu/Zn superoxide dismutase(Sod) mutant flies were accompanied by altered aging trajectories in motor circuit properties distinct from those in HT-reared flies, highlighting differential effects of oxidative versus temperature stressors. This work helps to identify several age-vulnerable neurophysiological parameters that may serve as quantitative indicators for assessing genetic and environmental influences on aging progression inDrosophila.

In 1963, Sidney Brenner wrote a proposal to Max Perutz on tackling the next ‘big questions’ in molecular biology. Beginning with the postulate that simple organisms have many of the features that c...

In the past, in the design of fracturing schemes, the influence of different factors on proppant-suspending ability of fracturing fluid was not considered comprehensively, resulting in incomplete matching between fracturing fluid and the requirements of proppant-suspending ability and incomplete fracturing schemes. In this paper, the effects of viscosities of clean fracturing fluid, proppant size and sand-liquid ratio on proppant-suspending ability at different temperatures were studied by physical simulation experiments in laboratory. The results show that the influence factors of proppant-suspending ability of fracturing fluid were listed in an effective order as below: viscosity of fracturing fluid, proppant size and sand-liquid ratio. The higher the viscosity of fracturing fluid is, the slower the settling rate of proppant is, and the stronger the proppant-suspending ability is. The larger the proppant size is, the faster the settling rate is, and the worse the proppant-suspending ability is. The time required for the settling to reach a stable state at different sand-liquid ratio is almost the same, which show that the proppant-suspending ability is almost the same. The higher the temperature is, the lower the viscosity of fracturing fluid is, the faster the settling rate is, and the worse the proppant-suspending ability is. Considering proppant-carrying ability, formation damage, economic cost and other factors, there is an optimum matching relationship between fracturing fluid viscosity and proppant particle size. Large size proppants need to suspend with high viscosity fracturing fluid, medium size proppants need to suspend with medium viscosity and above fracturing fluid, and small size proppants need to suspend with low viscosity and above fracturing fluid. The research results provide a basis for the optimization of fracturing schemes.

This study unravels the transcriptional response of a highly productive faba bean cultivar under vernalization treatment. Faba bean (Vicia faba L.) is a member of the Leguminosae family and an important food crop worldwide providing valuable nutrients for humans. However, genome-wide studies and comprehensive sequencing resources of faba bean remain limited. Vernalization is crucial for enhanced yields in a number of winter-sown crops. However, the effects of vernalization on faba bean remain unknown. In this study, we generated a high-quality transcriptome assembly and functional annotation source for vernalized faba bean (Vicia faba L.) cv. Tongxian-2, a domesticated cultivar from southern China. A total of 369.9 million clean Illumina paired-end RNA-Seq reads were generated, and the transcriptome was assembled into 68,683 unigene sequences, with an average length of 1018 bp and an N50 of 1652 bp. Comprehensive functional annotation provided putative functional descriptions for more than 70% of the faba bean transcripts. We annotated a total of 1560 faba bean transcripts encoding transcription factors (TFs) belonging to 55 distinct TF families. The bHLH (168 transcripts), ERF (123 transcripts) and WRKY (105 transcripts) contained the largest number of TFs in response to vernalization. Genome-wide transcript changes comparing vernalized and unvernalized seedlings were investigated using bioinformatics approaches, which revealed a strong repression of photosynthesis and carbon metabolism, while genes participating in 'response to stress' were significantly induced. We also specifically identified vernalization-induced twenty-two 'pollen-pistil interaction' genes. A detailed functional annotation and expression profile analyses unveiled a number of protein kinases, which were specifically induced in vernalized seedlings. We also identified a total of 6852 simple sequence repeats (SSRs) in 6552 transcripts, representing a valuable genomic molecular marker resource for faba bean. In summary, this study provides new insights into the vernalization process in this economically valuable crop. The transcriptome data obtained provides us with a valuable candidate gene resource for future functional and molecular breeding studies. These data will contribute to the genome annotation for ensuing genome projects.

The Drosophila mutant para(Shu) harbors a dominant, gain-of-function allele of the voltage-gated sodium channel gene, paralytic (para). The mutant flies display severe seizure-like phenotypes, including neuronal hyperexcitability, spontaneous spasms, ether-induced leg shaking, and heat-induced convulsions. We unexpectedly found that two distinct food recipes used routinely in the Drosophila research community result in a striking difference in severity of the para(Shu) phenotypes. Namely, when para(Shu) mutants were raised on the diet originally formulated by Edward Lewis in 1960, they showed severe neurological defects as previously reported. In contrast, when they were raised on the diet developed by Frankel and Brousseau in 1968, these phenotypes were substantially suppressed. Comparison of the effects of these two well-established food recipes revealed that the diet-dependent phenotypic suppression is accounted for by milk whey, which is present only in the latter. Inclusion of milk whey in the diet during larval stages was critical for suppression of the adult para(Shu) phenotypes, suggesting that this dietary modification affects development of the nervous system. We also found that milk whey has selective effects on other neurological mutants. Among the behavioral phenotypes of different para mutant alleles, those of para(GEFS+) and para(bss) were suppressed by milk whey, while those of para(DS) and para(ts1) were not significantly affected. Overall, our study demonstrates that different diets routinely used in Drosophila labs could have considerably different effects on neurological phenotypes of Drosophila mutants. This finding provides a solid foundation for further investigation into how dietary modifications affect development and function of the nervous system and, ultimately, how they influence behavior.

Intrinsic electric activities of neurons play important roles in establishing and refining neural circuits during development. However, how the underlying ionic currents undergo postembryonic reorganizations remains largely unknown. Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, we show drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK(Ca)) K+ currents during postembryonic development. Larval and adult neurons displayed prominent fast inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK(Ca) was far smaller in amplitude. In contrast, pupal neurons were characterized by large sustained IKv and prominent IK(Ca), encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removed inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents was accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolished the vast majority of IK(Ca), particularly at the pupal stage. Strikingly, the deficiency of IK(Ca) in slo pupae was compensated by the transient component of IKv mediated by Sh channels. Thus, IK(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survived normally, Sh;; slo double mutants deficient in both failed to survive through pupal metamorphosis. Together, our data highlight significant reorganizations and homeostatic compensations of K+ currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.

Figure 1. (A) Barry Ganetzky sitting on the ‘Steenbock Chair,’ photo by Jeff Miller, reprinted with permission from the University of Wisconsin-Madison. (B) Ganetzky giving his final remarks at the...

InDrosophila, high-frequency electrical stimulation across the brain triggers a highly stereotypic repertoire of spasms known as electroconvulsive seizures (ECS). The distinctive ECS spiking discharges manifest across the nervous system and can be stably assessed throughout the seizure repertoire in the large indirect flight muscles (DLMs). ECS discharges in DLMs have been extensively used to monitor seizure activities, notably in stress (‘bang’)-sensitive mutants. However, the relationships between ECS-spike patterns and native motor programs, including flight and grooming, are not known and their similarities and distinctions remain to be characterized. We employed quantitative spike pattern analyses including: 1) overall firing frequency, 2) spike timing between contralateral fibers, and 3) short-term variability in spike interval regularity (CV2) and instantaneous firing frequency (ISI−1) to reveal distinctions amongst ECS discharges, flight and grooming motor patterns. We then examined DLM firing phenotypes in well-established mutants in excitatory cholinergic (Cha), inhibitory GABAergic (Rdl) and electrical (ShakB) synaptic transmission. The results provide an initial glimpse on the vulnerability of individual motor patterns to perturbations of respective synaptic transmission systems. We found marked alterations of ECS discharge spike patterns in terms of either seizure threshold, spike frequency or spiking regularity. In contrast, no gross alterations during grooming and only a minor reduction of firing frequency during Rdl mutant flight were observed, suggesting a role for GABAergic modulation of flight motor programs. Picrotoxin (PTX), a known pro-convulsant that inhibits GABAAreceptors, induced DLM seizure patterns that displayed some features, e.g. left-right coordination and ISI−1range, that could be found in flight or grooming, but distinct from ECS discharges. Our results indicate that these quantitative techniques may be employed to reveal overlooked relationships among aberrant motor patterns and native DLM motor programs in genetic and pharmacological analyses of underlying cellular and neural circuit function.

GCaMP is an optogenetic Ca2+sensor widely used for monitoring neuronal activities but the precise physiological implications of GCaMP signals remain to be further delineated among functionally distinct synapses. TheDrosophilaneuromuscular junction (NMJ), a powerful genetic system for studying synaptic function and plasticity, consists of tonic and phasic glutamatergic and modulatory aminergic motor terminals of distinct properties. We report a first simultaneous imaging and electric recording study to directly contrast the frequency characteristics of GCaMP signals of the three synapses for physiological implications. Different GCaMP variants were applied in genetic and pharmacological perturbation experiments to examine the Ca2+influx and clearance processes underlying the GCaMP signal. Distinct mutational and drug effects on GCaMP signals indicate differential roles of Na+and K+channels, encoded by genes includingparalytic(para),Shaker(Sh),Shab, andether-a-go-go(eag), in excitability control of different motor terminals. Moreover, the Ca2+handling properties reflected by the characteristic frequency dependence of the synaptic GCaMP signals were determined to a large extent by differential capacity of mitochondria-powered Ca2+clearance mechanisms. Simultaneous focal recordings of synaptic activities further revealed that GCaMPs were ineffective in tracking the rapid dynamics of Ca2+influx that triggers transmitter release, especially during low-frequency activities, but more adequately reflected cytosolic residual Ca2+accumulation, a major factor governing activity-dependent synaptic plasticity. These results highlight the vast range of GCaMP response patterns in functionally distinct synaptic types and provide relevant information for establishing basic guidelines for the physiological interpretations of presynaptic GCaMP signals from in situ imaging studies.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder in humans characterized by complex behavioral deficits, including intellectual disability, impaired social interactions and hyperactivity. ASD exhibits a strong genetic component with underlying multi-gene interactions. Candidate gene studies have shown that the neurobeachin gene is disrupted in human patients with idiopathic autism (Castermans et al., 2003). The gene for neurobeachin (NBEA) spans the common fragile site FRA 13A and encodes a signal scaffold protein (Savelyeva et al., 2006). In mice, NBEA has been shown to be involved in the trafficking and function of a specific subset of synaptic vesicles. (Medrihan et al., 2009; Savelyeva, Sagulenko, Schmitt, & Schwab, 2006). rugose (rg) is the Drosophila homologue of the mammalian and human neurobeachin. Our previous genetic and molecular analyses have shown that rg encodes an A kinase anchor protein (DAKAP 550), which interacts with components of the EGFR and Notch mediated signaling pathways, facilitating cross-talk between these and other pathways (Shamloula et al., 2002). We now present functional data from studies on the larval neuromuscular junction that reveal abnormal synaptic architecture and physiology. In addition, adult rg loss-of-function mutants exhibit defective social interactions, impaired habituation, aberrant locomotion and hyperactivity. These results demonstrate that Drosophila neurobeachin (rugose) mutants exhibit phenotypic characteristics reminiscent of human ASD and thus could serve as a genetic model for studying autism spectrum disorders.

This special issue of the Journal of Neurogenetics honors the career of Professor Barry Ganetzky on the occasion of his retirement from the Laboratory of Genetics at the University of Wisconsin–Mad...

The Journal of Neurogenetics presents this special issue entitled “Neurogenetics of connectomes: from fly to fish.” In this special issue, we invited established researchers to review and present c...

Our earlier genetic screen uncovered a paraquat-sensitive leg-shaking mutant quiver1 (qvr1), whose gene product interacts with the Shaker (Sh) K+ channel (Humphreys et al, 1996; Wang et al., 2000). We also mapped the qvr locus to EY04063 and noticed altered day-night activity patterns in these mutants. Such circadian behavioral defects were independently reported by another group, who employed the qvr1 allele we supplied them, and attributed the extreme restless phenotype of EY04063 to the qvr gene. However, their report adopted a new noncanonical gene name sleepless (sss) for qvr (Koh et al., 2008). In addition to qvr1 and qvrEY, our continuous effort since the early 2000s generated a number of novel recessive qvr alleles, including EMS-induced mutations qvr2 and qvr3, and P-element excision lines qvrip6 (imprecise jumpout), qvrrv7 and qvrrv9 (revertants) derived from qvrEY. Distinct from the original intron-located qvr1 allele that generates abnormal-sized mRNAs, qvr2 and qvr3 had their lesion sites in exons 6 and 7, respectively, producing nearly normal-sized mRNA products. A set of RNA-editing sites are nearby the lesion sites of qvr3 and qvrEY on exon 7. Except for the revertants, all qvr alleles display a clear ether-induced leg-shaking phenotype just like Sh, and weakened climbing abilities to varying degrees. Unlike Sh, all shaking qvr alleles (except for qvrf01257) displayed a unique activity-dependent enhancement in excitatory junction potentials (EJPs) at larval neuromuscular junctions (NMJs) at very low stimulus frequencies, with qvrEY displaying the largest EJP and more significant NMJ overgrowth than other alleles. Our detailed characterization of a collection of qvr alleles help establish links between novel molecular lesions and different behavioral and physiological consequences, revealing how modifications of the qvr gene lead to a wide spectrum of phenotypes, including neuromuscular hyperexcitability, defective motor ability and activity-rest cycles.

The Journal of Neurogenetics presents this special issue entitled ‘Zebrafish: From Genes and Neurons to Circuits, Behavior and Disease’. We invited colleagues using the zebrafish model in the areas...

Tethered flies allow studies of biomechanics and electrophysiology of flight control. We performed microelectrode recordings of spikes in an indirect flight muscle (the dorsal longitudinal muscle, DLMa) coupled with acoustic analysis of wing beat frequency (WBF) via microphone signals. Simultaneous electrophysiological recording of direct and indirect flight muscles has been technically challenging; however, the WBF is thought to reflect in a one-to-one relationship with spiking activity in a subset of direct flight muscles, including muscle m1b. Therefore, our approach enables systematic mutational analysis for changes in temporal features of electrical activity of motor neurons innervating subsets of direct and indirect flight muscles. Here, we report the consequences of specific ion channel disruptions on the spiking activity of myogenic DLMs (firing at ∼5 Hz) and the corresponding WBF (∼200 Hz). We examined mutants of the genes enconding: 1) voltage-gated Ca(2+) channels (cacophony, cac), 2) Ca(2+)-activated K(+) channels (slowpoke, slo), and 3) voltage-gated K(+) channels (Shaker, Sh) and their auxiliary subunits (Hyperkinetic, Hk and quiver, qvr). We found flight initiation in response to an air puff was severely disrupted in both cac and slo mutants. However, once initiated, slo flight was largely unaltered, whereas cac displayed disrupted DLM firing rates and WBF. Sh, Hk, and qvr mutants were able to maintain normal DLM firing rates, despite increased WBF. Notably, defects in the auxiliary subunits encoded by Hk and qvr could lead to distinct consequences, that is, disrupted DLM firing rhythmicity, not observed in Sh. Our mutant analysis of direct and indirect flight muscle activities indicates that the two motor activity patterns may be independently modified by specific ion channel mutations, and that this approach can be extended to other dipteran species and additional motor programs, such as electroconvulsive stimulation-induced seizures.

Recent analyses in flies, mice, zebrafish, and humans showed that mutations in prickle orthologs result in epileptic phenotypes, although the mechanism responsible for generating the seizures was unknown. Here, we show that Prickle organizes microtubule polarity and affects their growth dynamics in axons of Drosophila neurons, which in turn influences both anterograde and retrograde vesicle transport. We also show that enhancement of the anterograde transport mechanism is the cause of the seizure phenotype in flies, which can be suppressed by reducing the level of either of two Kinesin motor proteins responsible for anterograde vesicle transport. Additionally, we show that seizure-prone prickle mutant flies have electrophysiological defects similar to other fly mutants used to study seizures, and that merely altering the balance of the two adult prickle isoforms in neurons can predispose flies to seizures. These data reveal a previously unidentified pathway in the pathophysiology of seizure disorders and provide evidence for a more generalized cellular mechanism whereby Prickle mediates polarity by influencing microtubule-mediated transport.

Pyridox(am)ine 5’-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting dietary vitamin B6 (VB6) to pyridoxal 5’-phosphate (PLP), the biologically active form of VB6, and involved in the synthesis of neuro-transmitters including GABA, dopamine, and serotonin. In humans, PNPO mutations have been increasingly identified in neonatal epileptic encephalopathy and more recently also in early-onset epilepsy. Till now, little is known about the neurobiological mechanisms underlying PNPO-deficiency-induced seizures due to the lack of animal models. Previously we identified a c.95 C > A missense mutation insgll- theDrosophilahomolog of humanPNPO(hPNPO) and found mutant (sgll95) flies exhibiting a lethal phenotype on a diet devoid of VB6. Here we report the establishment of bothsgll95and ubiquitoussgllknockdown (KD) flies as valid animal models of PNPO-deficiency-induced epilepsy. Bothsgll95andsgllKD flies exhibit spontaneous seizures before they die. Electrophysiological recordings reveal that seizures caused by PNPO deficiency have characteristics similar to that in flies treated with GABA antagonist picrotoxin. Both seizures and lethality are associated with low PLP levels and can be rescued by ubiquitous expression of wild-typesgllorhPNPO, suggesting the functional conservation of the PNPO enzyme between humans and flies. Results from cell type-specificsgllKD further demonstrate that PNPO in the brain is necessary for seizure prevention and survival. Our establishment of the first animal model of PNPO deficiency will lead to better understanding of VB6 biology, thePNPOgene and its mutations discovered in patients, and can be a cost-effective system to test therapeutic strategies.

Ca2+ influx through voltage-activated Ca2+ channels and its feedback regulation by Ca2+-activated K+ (BK) channels is critical in Ca2+-dependent cellular processes, including synaptic transmission, growth and homeostasis. Here we report differential roles of cacophony (CaV2) and Dmca1D (CaV1) Ca2+ channels in synaptic transmission and in synaptic homeostatic regulations induced by slowpoke (slo) BK channel mutations. At Drosophila larval neuromuscular junctions (NMJs), a well-established homeostatic mechanism of transmitter release enhancement is triggered by experimentally suppressing postsynaptic receptor response. In contrast, a distinct homeostatic adjustment is induced by slo mutations. To compensate for the loss of BK channel control presynaptic Sh K+ current is upregulated to suppress transmitter release, coupled with a reduction in quantal size. We demonstrate contrasting effects of cac and Dmca1D channels in decreasing transmitter release and muscle excitability, respectively, consistent with their predominant pre- vs. postsynaptic localization. Antibody staining indicated reduced postsynaptic GluRII receptor subunit density and altered ratio of GluRII A and B subunits in slo NMJs, leading to quantal size reduction. Such slo-triggered modifications were suppressed in cac;;slo larvae, correlated with a quantal size reversion to normal in double mutants, indicating a role of cac Ca2+ channels in slo-triggered homeostatic processes. In Dmca1D;slo double mutants, the quantal size and quantal content were not drastically different from those of slo, although Dmca1D suppressed the slo-induced satellite bouton overgrowth. Taken together, cac and Dmca1D Ca2+ channels differentially contribute to functional and structural aspects of slo-induced synaptic modifications. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 1–15, 2014

This paper analyzes the side space’s impact on deformation and stress of the inner pressure tank shell under different load cases, depending on the stress law of the outer pressure tank. The results indicate that: the smaller side space is good for the longitudinal stress at the shell above the pressure tank under two conditions. As the side space increases, the stress decreases. The stress’s changing law of the inner pressure tank’s shell is the same with the outer. However, the changing law of the hull’s stress is different. The stress computational formula of the outer pressure tank can’t be applied to the inner directly and it need be modified.

Shudderer (Shu) is an X-linked dominant mutation in Drosophila melanogaster identified more than 40 years ago. A previous study showed that Shu caused spontaneous tremors and defects in reactive climbing behavior, and that these phenotypes were significantly suppressed when mutants were fed food containing lithium, a mood stabilizer used in the treatment of bipolar disorder (Williamson, 1982). This unique observation suggested that the Shu mutation affects genes involved in lithium-responsive neurobiological processes. In the present study, we identified Shu as a novel mutant allele of the voltage-gated sodium (Nav) channel gene paralytic (para). Given that hypomorphic para alleles and RNA interference–mediated para knockdown reduced the severity of Shu phenotypes, Shu was classified as a para hypermorphic allele. We also demonstrated that lithium could improve the behavioral abnormalities displayed by other Nav mutants, including a fly model of the human generalized epilepsy with febrile seizures plus. Our electrophysiological analysis of Shu showed that lithium treatment did not acutely suppress Nav channel activity, indicating that the rescue effect of lithium resulted from chronic physiological adjustments to this drug. Microarray analysis revealed that lithium significantly alters the expression of various genes in Shu, including those involved in innate immune responses, amino acid metabolism, and oxidation-reduction processes, raising the interesting possibility that lithium-induced modulation of these biological pathways may contribute to such adjustments. Overall, our findings demonstrate that Nav channel mutants in Drosophila are valuable genetic tools for elucidating the effects of lithium on the nervous system in the context of neurophysiology and behavior.