Intense light unleashes male-male courtship behaviour in wild-type Drosophila.

Drosophila courtship studies have elucidated several principles of the neurogenetic organization of complex behaviour. 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 behaviours among male flies. Strikingly, in wild-type male-only chambers, we observed extreme behavioural manifestations, such as 'chaining' and 'wheeling', resembling previously reported male-male courtship behaviours in fruitless mutants and in transformants with ectopic mini-white+ overexpression. This male-male courtship was greatly diminished in a variety of visual system mutants, including disrupted phototransduction (norpA), eliminated eye-colour 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 mutants orco and sbl displayed unrestrained male-male courtship. Particularly, orco males attained maximum courtship scores under either dim or intense light conditions. Together, our observations support the notion that the innate male courtship behaviour is restrained by olfactory cues under normal conditions but can be unleashed by strong visual stimulation in Drosophila.

Abnormal larval neuromuscular junction morphology and physiology in Drosophila prickle isoform mutants with known axonal transport defects and adult seizure behavior.

Previous studies have demonstrated the striking mutational effects of the Drosophila planar cell polarity gene prickle (pk) on larval motor axon microtubule-mediated vesicular transport and on adult epileptic behavior associated with neuronal circuit hyperexcitability. Mutant alleles of the prickle-prickle (pkpk) and prickle-spiny-legs (pksple) isoforms (hereafter referred to as pk and sple alleles, respectively) exhibit differential phenotypes. While both pk and sple affect larval motor axon transport, only sple confers motor circuit and behavior hyperexcitability. However, mutations in the two isoforms apparently counteract to ameliorate adult motor circuit and behavioral hyperexcitability in heteroallelic pkpk/pksple flies. We have further investigated the consequences of altered axonal transport in the development and function of the larval neuromuscular junction (NMJ). We uncovered robust dominant phenotypes in both pk and sple alleles, including synaptic terminal overgrowth (as revealed by anti-HRP and -Dlg immunostaining) and poor vesicle release synchronicity (as indicated by synaptic bouton focal recording). However, we observed recessive alteration of synaptic transmission only in pk/pk larvae, i.e. increased excitatory junctional potential (EJP) amplitude in pk/pk but not in pk/+ or sple/sple. Interestingly, for motor terminal excitability sustained by presynaptic Ca2+ channels, both pk and sple exerted strong effects to produce prolonged depolarization. Notably, only sple acted dominantly whereas pk/+ appeared normal, but was able to suppress the sple phenotypes, i.e. pk/sple appeared normal. Our observations contrast the differential roles of the pk and sple isoforms and highlight their distinct, variable phenotypic expression in the various structural and functional aspects of the larval NMJ.

Drosophila carrying epilepsy-associated variants in the vitamin B6 metabolism gene PNPO display allele- and diet-dependent phenotypes.

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 gamma-aminobutyric acid (GABA) and the monoamines. Pathogenic variants in PNPO have been increasingly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities. Recently, the PNPO gene has also been implicated in epilepsy in adults. It is unclear how these phenotypic variations are linked to specific PNPO alleles and to what degree diet can modify their expression. Using CRISPR-Cas9, we generated four knock-in Drosophila alleles, hWT , hR116Q , hD33V , and hR95H , in which the endogenous Drosophila PNPO was replaced by wild-type human PNPO complementary DNA (cDNA) and three epilepsy-associated variants. We found that these knock-in flies exhibited a wide range of phenotypes, including developmental impairments, abnormal locomotor activities, spontaneous seizures, and shortened life span. These phenotypes are allele dependent, varying with the known biochemical severity of these mutations and our characterized molecular defects. We also showed that diet treatments further diversified the phenotypes among alleles, and PLP supplementation at larval and adult stages prevented developmental impairments and seizures in adult flies, respectively. Furthermore, we found that hR95H had a significant dominant-negative effect, rendering heterozygous flies susceptible to seizures and premature death. Together, these results provide biological bases for the various phenotypes resulting from multifunction of PNPO, specific molecular and/or genetic properties of each PNPO variant, and differential allele-diet interactions.

Distinct Aging-Vulnerable and -Resilient Trajectories of Specific Motor Circuit Functions in Oxidation- and Temperature-Stressed Drosophila.

In Drosophila, 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 in Drosophila across 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 expediting Drosophila aging studies. We discovered that shortened life spans in Cu/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 in Drosophila.

Fly seizure EEG: field potential activity in the Drosophila brain.

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.

Differential effects on neuromuscular physiology between Sod1 loss-of-function mutation and paraquat-induced oxidative stress in Drosophila.

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.

Comprehensive transcriptome analysis of faba bean in response to vernalization.

MAIN CONCLUSION: 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.

Pyridox (am) ine 5'-phosphate oxidase deficiency induces seizures in Drosophila melanogaster.

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 C>A 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.

Milk-whey diet substantially suppresses seizure-like phenotypes of paraShu, a Drosophila voltage-gated sodium channel mutant.

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, 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 paraShu phenotypes. Namely, when paraShu 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 paraShu 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 paraGEFS+ and parabss were suppressed by milk whey, while those of paraDS and parats1 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.

Distinctions among electroconvulsion- and proconvulsant-induced seizure discharges and native motor patterns during flight and grooming: quantitative spike pattern analysis in Drosophila flight muscles.

In Drosophila, high-frequency electrical stimulation across the brain triggers a highly stereotypic repertoire of spasms. These electroconvulsive seizures (ECS) manifest as distinctive spiking discharges across the nervous system and can be stably assessed throughout the seizure repertoire in the large indirect flight muscles dorsal longitudinal muscles (DLMs) to characterize modifications in seizure-prone 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 for the three motor patterns 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). This base-line information from wild-type (WT) flies facilitated quantitative characterization of mutational effects of major neurotransmitter systems: 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 different perturbations. 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 a small but noticeable reduction of firing frequency during Rdl mutant flight were found, suggesting a role for GABAergic modulation of flight motor programs. Picrotoxin (PTX), a known pro-convulsant that inhibits GABAA receptors, induced DLM spike patterns that displayed some features, e.g. left-right coordination and ISI-1 range, that could be found in flight or grooming, but distinct from ECS discharges. These quantitative techniques may be employed to reveal overlooked relationships among aberrant motor patterns as well as their links to native motor programs.

Inter-relationships among physical dimensions, distal-proximal rank orders, and basal GCaMP fluorescence levels in Ca2+ imaging of functionally distinct synaptic boutons at Drosophila neuromuscular junctions.

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). We investigated whether variability in synaptic bouton sizes and GCaMP expression levels confound interpretations of GCaMP readouts, in inferring the intrinsic Ca2+ handling properties among these functionally distinct synapses. Analysis of large data sets accumulated over years established the wide ranges of bouton sizes and GCaMP baseline fluorescence, with large overlaps among synaptic categories. We showed that bouton size and GCaMP baseline fluorescence were not confounding factors in determining the characteristic frequency responses among type Ib, Is and II synapses. More importantly, the drastic phenotypes that hyperexcitability mutations manifest preferentially in particular synaptic categories, were not obscured by bouton heterogeneity in physical size and GCaMP expression level. Our data enabled an extensive analysis of the distal-proximal gradient of GCaMP responses upon genetic and pharmacological manipulations. The results illustrate the conditions that disrupt or enhance the distal-proximal gradients. For example, stimulus frequencies just above the threshold level produced the steepest gradient in low Ca2+ (0.1 mM) saline, while supra-threshold stimulation flattened the gradient. Moreover, membrane hyperexcitability mutations (eag1 Sh120 and parabss1) and mitochondrial inhibition by dinitrophenol (DNP) disrupted the gradient. However, a novel distal-proximal gradient of decay kinetics appeared after long-term DNP incubation. We performed focal recording to assess the failure rates in transmission at low Ca2+ levels, which yielded indications of a mild distal-proximal gradient in release probability.

Unraveling Synaptic GCaMP Signals: Differential Excitability and Clearance Mechanisms Underlying Distinct Ca2+ Dynamics in Tonic and Phasic Excitatory, and Aminergic Modulatory Motor Terminals in Drosophila.

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. The Drosophila neuromuscular 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 including paralytic (para), Shaker (Sh), Shab, and ether-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.