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Developing neural circuits are influenced by activity and are especially sensitive to changes in activity during critical periods (CPs) of development. Changes occurring during a CP often become 'locked in' so that they affect the mature network. Indeed, several neurodevelopmental disorders have been linked to excessive activity during such periods. It is, therefore, important to identify those aspects of neural circuit development that are influenced by neural activity during a CP. In this study, we take advantage of the genetic tractability of Drosophila to show that activity perturbation during an embryonic CP permanently alters properties of the locomotor circuit. Specific changes we identify include increased synchronicity of motoneuron activity and greater strengthening of excitatory over inhibitory synaptic drive to motoneurons. These changes are sufficient to reduce network robustness, evidenced by increased sensitivity to induced seizure. We also show that we can rescue these changes when increased activity is mitigated by inhibition provided by mechanosensory neurons. Similarly, we demonstrate a dose-dependent relationship between inhibition experienced during the CP and the extent to which it is possible to rescue the hyperexcitable phenotype characteristic of the para bss mutation. This suggests that developing circuits must be exposed to a properly balanced sum of excitation and inhibition during the CP to achieve normal mature network function. Our results, therefore, provide novel insight into how activity during a CP shapes specific elements of a circuit, and how activity during this period is integrated to tune neural circuits to the environment in which they will likely function., Competing Interests: IH, BC, TP, JD, JP, ML, RB No competing interests declared, (© 2023, Hunter, Coulson et al.)

Central nervous system organogenesis is a complex process that obeys precise architectural rules. The impact that nervous system architecture may have on its functionality remains, however, relatively unexplored. To clarify this problem, we analyze the development of the Drosophila embryonic Ventral Nerve Cord (VNC). VNC morphogenesis requires the tight control of Jun kinase (JNK) signaling in a subset of pioneer neurons, exerted in part via a negative feedback loop mediated by the dual specificity phosphatase Puckered. Here we show that the JNK pathway autonomously regulates neuronal electrophysiological properties without affecting synaptic vesicle transport. Manipulating JNK signaling activity in pioneer neurons during early embryogenesis directly influences their function as organizers of VNC architecture and, moreover, uncovers a role in the coordination of the embryonic motor circuitry that is required for hatching. Together, our data reveal critical links, mediated by the control of the JNK signaling cascade by Puckered, between the structural organization of the VNC and its functional optimization., (© 2023. The Author(s).)

Autism spectrum disorder (ASD) is a common neurodevelopmental condition for which there are no pharmacological therapies that effectively target its core symptomatology. Animal models of syndromic forms of ASD, such as neurofibromatosis type 1, may be of use in screening for such treatments. Drosophila larvae lacking Nf1 expression exhibit tactile hypersensitivity following mechanical stimulation, proposed to mirror the sensory sensitivity issues comprising part of the ASD diagnostic criteria. Such behavior is associated with synaptic dysfunction at the neuromuscular junction (NMJ). Both phenotypes may thus provide tractable outputs with which to screen for potential ASD therapies. In this study, we demonstrate that, while loss of Nf1 expression within the embryo is sufficient to impair NMJ synaptic transmission in the larva, constitutive Nf1 knock-down is required to induce tactile hypersensitivity, suggesting that a compound must be administered throughout development to rescue this behavior. With such a feeding regime, we identify two compounds from a targeted, low-throughput screen that significantly and consistently reduce, but do not fully rescue, tactile hypersensitivity in Nf1 P1 larvae. These are the HMG CoA-reductase inhibitor simvastatin, and the BK Ca channel activator BMS-204352. At the NMJ, both compounds induce a significant reduction in the enhanced spontaneous transmission frequency of Nf1 P1 larvae, though again not to the level of vehicle-treated controls. However, both compounds fully rescue the increased quantal size of Nf1 P1 mutants, with simvastatin also fully rescuing their reduced quantal content. Thus, the further study of both compounds as potential ASD interventions is warranted., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Dyson et al.)

Pathogenic, generally loss-of-function, variants in CACNA1F , encoding the Ca v 1.4α 1 calcium channel, underlie congenital stationary night blindness type 2 (CSNB2), a rare inherited retinal disorder associated with visual disability. To establish the underlying pathomechanism, we investigated 10 clinically derived CACNA1F missense variants located across pore-forming domains, connecting loops, and the carboxy-tail domain of the Ca v 1.4α subunit. Homology modeling showed that all variants cause steric clashes; informatics analysis correctly predicted pathogenicity for 7/10 variants. In vitro analyses demonstrated that all variants cause a decrease in current, global expression, and protein stability and act through a loss-of-function mechanism and suggested that the mutant Ca v 1.4α proteins were degraded by the proteasome. We showed that the reduced current for these variants could be significantly increased through treatment with clinical proteasome inhibitors. In addition to facilitating clinical interpretation, these studies suggest that proteasomal inhibition represents an avenue of potential therapeutic intervention for CSNB2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sadeh, Baines, Black and Manson.)

The vacuolar H+-ATPase is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. We describe heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the vacuolar H+-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modelling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased vacuolar H+-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behaviour, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder and provides insight into disease mechanisms., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Many animals use Earth's magnetic field (also known as the geomagnetic field) for navigation 1 . The favoured mechanism for magnetosensitivity involves a blue-light-activated electron-transfer reaction between flavin adenine dinucleotide (FAD) and a chain of tryptophan residues within the photoreceptor protein CRYPTOCHROME (CRY). The spin-state of the resultant radical pair, and therefore the concentration of CRY in its active state, is influenced by the geomagnetic field 2 . However, the canonical CRY-centric radical-pair mechanism does not explain many physiological and behavioural observations 2-8 . Here, using electrophysiology and behavioural analyses, we assay magnetic-field responses at the single-neuron and organismal levels. We show that the 52 C-terminal amino acid residues of Drosophila melanogaster CRY, lacking the canonical FAD-binding domain and tryptophan chain, are sufficient to facilitate magnetoreception. We also show that increasing intracellular FAD potentiates both blue-light-induced and magnetic-field-dependent effects on the activity mediated by the C terminus. High levels of FAD alone are sufficient to cause blue-light neuronal sensitivity and, notably, the potentiation of this response in the co-presence of a magnetic field. These results reveal the essential components of a primary magnetoreceptor in flies, providing strong evidence that non-canonical (that is, non-CRY-dependent) radical pairs can elicit magnetic-field responses in cells., (© 2023. The Author(s).)

Autism spectrum disorder (ASD) is a neurodevelopmental condition in which the mechanisms underlying its core symptomatology are largely unknown. Studying animal models of monogenic syndromes associated with ASD, such as neurofibromatosis type 1 (NF1), can offer insights into its etiology. Here, we show that loss of function of the Drosophila NF1 ortholog results in tactile hypersensitivity following brief mechanical stimulation in the larva (mixed sexes), paralleling the sensory abnormalities observed in individuals with ASD. Mutant larvae also exhibit synaptic transmission deficits at the glutamatergic neuromuscular junction (NMJ), with increased spontaneous but reduced evoked release. While the latter is homeostatically compensated for by a postsynaptic increase in input resistance, the former is consistent with neuronal hyperexcitability. Indeed, diminished expression of NF1 specifically within central cholinergic neurons induces both excessive neuronal firing and tactile hypersensitivity, suggesting the two may be linked. Furthermore, both impaired synaptic transmission and behavioral deficits are fully rescued via knock-down of Ras proteins. These findings validate NF1 -/- Drosophila as a tractable model of ASD with the potential to elucidate important pathophysiological mechanisms. SIGNIFICANCE STATEMENT Autism spectrum disorder (ASD) affects 1-2% of the overall population and can considerably impact an individual's quality of life. However, there are currently no treatments available for its core symptoms, largely because of a poor understanding of the underlying mechanisms involved. Examining how loss of function of the ASD-associated NF1 gene affects behavior and physiology in Drosophila may shed light on this. In this study, we identify a novel, ASD-relevant behavioral phenotype in NF1 -/- larvae, namely an enhanced response to mechanical stimulation, which is associated with Ras-dependent synaptic transmission deficits indicative of neuronal hyperexcitability. Such insights support the use of Drosophila neurofibromatosis type 1 (NF1) models in ASD research and may provide outputs for genetic or pharmacological screens in future studies., (Copyright © 2022 the authors.)

Critical periods are phases of heightened plasticity that occur during the development of neural networks. Beginning with pioneering work of Hubel and Wiesel, which identified a critical period for the formation of ocular dominance in mammalian visual network connectivity, critical periods have been identified for many circuits, both sensory and motor, and across phyla, suggesting a universal phenomenon. However, a key unanswered question remains why these forms of plasticity are restricted to specific developmental periods rather than being continuously present. The consequence of this temporal restriction is that activity perturbations during critical periods can have lasting and significant functional consequences for mature neural networks. From a developmental perspective, critical period plasticity might enable reproducibly robust network function to emerge from ensembles of cells, whose properties are necessarily variable and fluctuating. Critical periods also offer significant clinical opportunity. Imposed activity perturbation during these periods has shown remarkable beneficial outcomes in a range of animal models of neurological disease including epilepsy. In this review, we spotlight the recent identification of a locomotor critical period in Drosophila larva and describe how studying this model organism, because of its simplified nervous system and an almost complete wired connectome, offers an attractive prospect of understanding how activity during a critical period impacts a neuronal network., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Coulson, Hunter, Doran, Parkin, Landgraf and Baines.)

Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The Drosophila connectome project aims to map the synaptic connectivity of entire larval and adult fly neural networks, which is essential for understanding nervous system development and function. So far, the project has produced an impressive amount of electron microscopy data that has facilitated reconstructions of specific synapses, including many in the larval locomotor circuit. While this breakthrough represents a technical tour de force, the data remain underutilized, partly because of a lack of functional validation of reconstructions. Attempts to validate connectivity posited by the connectome project, have mostly relied on behavioral assays and/or GFP reconstitution across synaptic partners (GRASP) or GCaMP imaging. While these techniques are useful, they have limited spatial or temporal resolution. Electrophysiological assays of synaptic connectivity overcome these limitations. Here, we combine patch-clamp recordings with optogenetic stimulation in male and female larvae, to test synaptic connectivity proposed by connectome reconstructions. Specifically, we use multiple driver lines to confirm that several connections between premotor interneurons and the anterior corner cell motoneuron are, as the connectome project suggests, monosynaptic. In contrast, our results also show that conclusions based on GRASP imaging may provide false-positive results regarding connectivity between cells. We also present a novel imaging tool, based on the same technology as our electrophysiology, as a favorable alternative to GRASP imaging. Finally, of eight Gal4 lines tested, five are reliably expressed in the premotor interneurons they are targeted to. Thus, our work highlights the need to confirm functional synaptic connectivity, driver line specificity, and use of appropriate genetic tools to support connectome projects. SIGNIFICANCE STATEMENT The Drosophila connectome project aims to provide a complete description of connectivity between neurons in an organism that presents experimental advantages over other models. It has reconstructed hundreds of thousands of synaptic connections of the fly larva by manual identification of anatomic landmarks present in serial section transmission electron microscopy (ssTEM) volumes of the larval CNS. We use a highly reliable electrophysiological approach to verify these connections, providing useful insight into the accuracy of work based on ssTEM. We also present a novel imaging tool for validating excitatory monosynaptic connections between cells and show that several genetic driver lines designed to target neurons of the larval connectome exhibit nonspecific and/or unreliable expression., (Copyright © 2022 the authors.)

Developing nervous systems exhibit spontaneous activity. Two new studies identify the instructive influence such activity has on the formation of functionally appropriate circuits., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. We have made a knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, we performed an NMR metabolomic profiling of heads, which revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments.

The emergence of coordinated network function during nervous system development is often associated with critical periods. These phases are sensitive to activity perturbations during, but not outside, of the critical period, that can lead to permanently altered network function for reasons that are not well understood. In particular, the mechanisms that transduce neuronal activity to regulating changes in neuronal physiology or structure are not known. Here, we take advantage of a recently identified invertebrate model for studying critical periods, the Drosophila larval locomotor system. Manipulation of neuronal activity during this critical period is sufficient to increase synaptic excitation and to permanently leave the locomotor network prone to induced seizures. Using genetics and pharmacological manipulations, we identify nitric oxide (NO)-signaling as a key mediator of activity. Transiently increasing or decreasing NO-signaling during the critical period mimics the effects of activity manipulations, causing the same lasting changes in synaptic transmission and susceptibility to seizure induction. Moreover, the effects of increased activity on the developing network are suppressed by concomitant reduction in NO-signaling and enhanced by additional NO-signaling. These data identify NO signaling as a downstream effector, providing new mechanistic insight into how activity during a critical period tunes a developing network., (© 2021. The Author(s).)

Epilepsy is one of the most common neurologic disorders. Around one third of patients do not respond to current medications. This lack of treatment indicates a need for better understanding of the underlying mechanisms and, importantly, the identification of novel targets for drug manipulation. The fruit fly Drosophila melanogaster has a fast reproduction time, powerful genetics, and facilitates large sample sizes, making it a strong model of seizure mechanisms. To better understand behavioral and physiological phenotypes across major fly seizure genotypes we systematically measured seizure severity and secondary behavioral phenotypes at both the larval and adult stage. Comparison of several seizure-induction methods; specifically electrical, mechanical and heat induction, show that larval electroshock is the most effective at inducing seizures across a wide range of seizure-prone mutants tested. Locomotion in adults and larvae was found to be non-predictive of seizure susceptibility. Recording activity in identified larval motor neurons revealed variations in action potential (AP) patterns, across different genotypes, but these patterns did not correlate with seizure susceptibility. To conclude, while there is wide variation in mechanical induction, heat induction, and secondary phenotypes, electroshock is the most consistent method of seizure induction across known major seizure genotypes in Drosophila ., (Copyright © 2021 Mituzaite et al.)

Primary cilia are compartmentalised from the rest of the cell by a ciliary gate comprising transition fibres and a transition zone. The ciliary gate allows the selective import and export of molecules such as transmembrane receptors and transport proteins. These are required for the assembly of the cilium, its function as a sensory and signalling centre and to maintain its distinctive composition. Certain motile cilia can also form within the cytosol as exemplified by human and Drosophila sperm. The role of transition fibre proteins has not been well described in the cytoplasmic cilia. Drosophila have both compartmentalised primary cilia, in sensory neurons, and sperm flagella that form within the cytosol. Here, we describe phenotypes for twitchy the Drosophila orthologue of a transition fibre protein, mammalian FBF1/C. elegans dyf-19. Loss-of-function mutants in twitchy are adult lethal and display a severely uncoordinated phenotype. Twitchy flies are too uncoordinated to mate but RNAi-mediated loss of twitchy specifically within the male germline results in coordinated but infertile adults. Examination of sperm from twitchy RNAi-knockdown flies shows that the flagellar axoneme forms, elongates and is post-translationally modified by polyglycylation but the production of motile sperm is impaired. These results indicate that twitchy is required for the function of both sensory cilia that are compartmentalised from the rest of the cell and sperm flagella that are formed within the cytosol of the cell. Twitchy is therefore likely to function as part of a molecular gate in sensory neurons but may have a distinct function in sperm cells., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

It is difficult to answer important questions in neuroscience, such as: "how do neural circuits generate behaviour?," because research is limited by the complexity and inaccessibility of the mammalian nervous system. Invertebrate model organisms offer simpler networks that are easier to manipulate. As a result, much of what we know about the development of neural circuits is derived from work in crustaceans, nematode worms and arguably most of all, the fruit fly, Drosophila melanogaster . This review aims to demonstrate the utility of the Drosophila larval locomotor network as a model circuit, to those who do not usually use the fly in their work. This utility is explored first by discussion of the relatively complete connectome associated with one identified interneuron of the locomotor circuit, A27h, and relating it to similar circuits in mammals. Next, it is developed by examining its application to study two important areas of neuroscience research: critical periods of development and interindividual variability in neural circuits. In summary, this article highlights the potential to use the larval locomotor network as a "generic" model circuit, to provide insight into mammalian circuit development and function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hunter, Coulson, Zarin and Baines.)

A large intronic hexanucleotide repeat expansion (GGGGCC) within the C9orf72 (C9orf72-SMCR8 Complex Subunit) locus is the most prevalent genetic cause of both Frontotemporal Dementia (FTD) and Motor Neuron Disease (MND). In patients this expansion is typically hundreds to thousands of repeat units in length. Repeat associated non-AUG translation of the expansion leads to the formation of toxic, pathological Dipeptide-Repeat Proteins (DPRs). To date there remains a lack of in vivo models expressing C9orf72 related DPRs with a repeat length of more than a few hundred repeats. As such our understanding of how physiologically relevant repeat length DPRs effect the nervous system in an ageing in vivo system remains limited. In this study we generated Drosophila models expressing DPRs over 1000 repeat units in length, a known pathological length in humans. Using these models, we demonstrate each DPR exhibits a unique, age-dependent, phenotypic and pathological profile. Furthermore, we show co-expression of specific DPR combinations leads to distinct, age-dependent, phenotypes not observed through expression of single DPRs. We propose these models represent a unique, in vivo, tool for dissecting the molecular mechanisms implicated in disease pathology, opening up new avenues in the study of both MND and FTD.

Voltage-gated sodium channels (VGSCs) are a major target site for the action of pyrethroid insecticides and resistance to pyrethroids has been ascribed to mutations in the VGSC gene. VGSCs in insects are encoded by only one gene and their structural and functional diversity results from posttranscriptional modification, particularly, alternative splicing. Using whole cell patch clamping of neurons from pyrethroid susceptible (wild-type) and resistant strains (s-kdr) of housefly, Musca domestica, we have shown that the V 50 for activation and steady state inactivation of sodium currents (I Na+ ) is significantly depolarised in s-kdr neurons compared with wild-type and that 10 nM deltamethrin significantly hyperpolarised both of these parameters in the neurons from susceptible but not s-kdr houseflies. Similarly, tail currents were more sensitive to deltamethrin in wild-type neurons (EC 15 14.5 nM) than s-kdr (EC 15 133 nM). We also found that in both strains, I Na+ are of two types: a strongly inactivating (to 6.8% of peak) current, and a more persistent (to 17.1% of peak) current. Analysis of tail currents showed that the persistent current in both strains (wild-type EC 15 5.84 nM) was more sensitive to deltamethrin than was the inactivating type (wild-type EC 15 35.1 nM). It has been shown previously, that the presence of exon l in the Drosophila melanogaster VGSC gives rise to a more persistent I Na+ than does the alternative splice variant containing exon k and we used PCR with housefly head cDNA to confirm the presence of the housefly orthologues of splice variants k and l. Their effect on deltamethrin sensitivity was determined by examining I Na+ in Xenopus oocytes expressing either the k or l variants of the Drosophila para VGSC. Analysis of tail currents, in the presence of various concentrations of deltamethrin, showed that the l splice variant was significantly more sensitive (EC 50 42 nM) than the k splice variant (EC 50 866 nM). We conclude that in addition to the presence of point mutations, target site resistance to pyrethroids may involve the differential expression of splice variants., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl- channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy., Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp., Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1., Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration-approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment 1 . In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes 1 . Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis.

Pumilio (Pum), an RNA-binding protein, is a key component of neuron firing-rate homeostasis that likely maintains stability of neural circuit activity in all animals, from flies to mammals. While Pum is ubiquitously expressed, we understand little about how synaptic excitation regulates its expression in the CNS. Here, we characterized the Drosophila dpum promoter and identified multiple myocyte enhancer factor-2 (Mef2)-binding elements. We cloned 12 dmef2 splice variants and used a luciferase-based assay to monitor dpum promoter activity. While all 12 dMef2 splice variants enhance dpum promoter activity, exon 10-containing variants induce greater transactivation. Previous work shows dPum expression increases with synaptic excitation. However, we observe no change in dmef2 transcript in larval CNS, of both sexes, exposed to the proconvulsant picrotoxin. The lack of activity dependence is indicative of additional regulation. We identified p300 as a potential candidate. We show that by binding to dMef2, p300 represses dpum transactivation. Significantly, p300 transcript is downregulated by enhanced synaptic excitation (picrotoxin) which, in turn, increases transcription of dpum through derepression of dMef2. These results advance our understanding of dpum by showing the activity-dependent expression is regulated by an interaction between p300 and dMef2., (© 2019 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The Q-system is a binary expression system that works well across species. Here, we report the development and demonstrate the applications of a split-QF system that drives strong expression in Drosophila , is repressible by QS, and is inducible by a small nontoxic molecule (quinic acid). The split-QF system is fully compatible with existing split-GAL4 and split-LexA lines, thus greatly expanding the range of possible advanced intersectional experiments and anatomical, physiological, and behavioral assays in Drosophila , and in other organisms., (Copyright © 2019 Riabinina et al.)

While the primary role of vesicular transporters is to load neurotransmitters into synaptic vesicles (SVs), accumulating evidence suggests that these proteins also contribute to additional aspects of synaptic function, including vesicle release. In this study, we extend the role of the VAChT to include regulating the transmitter content of SVs. We report that manipulation of a C-terminal poly-glutamine (polyQ) region in the Drosophila VAChT is sufficient to influence transmitter content, and release frequency, of cholinergic vesicles from the terminals of premotor interneurons. Specifically, we find that reduction of the polyQ region, by one glutamine residue ( 13Q to 12Q ), results in a significant increase in both amplitude and frequency of spontaneous cholinergic miniature EPSCs (mEPSCs) recorded in the aCC and RP2 motoneurons. Moreover, this truncation also results in evoked synaptic currents that show increased duration: consistent with increased ACh release. By contrast, extension of the polyQ region by one glutamine ( 13Q to 14Q ) is sufficient to reduce mEPSC amplitude and frequency and, moreover, prevents evoked SV release. Finally, a complete deletion of the polyQ region (13Q to 0Q) has no obvious effects to mEPSCs, but again evoked synaptic currents show increased duration. The mechanisms that ensure SVs are filled to physiologically-appropriate levels remain unknown. Our study identifies the polyQ region of the insect VAChT to be required for correct vesicle transmitter loading and, thus, provides opportunity to increase understanding of this critical aspect of neurotransmission., Competing Interests: Conflict of Interest Statement: S.W.V. and J.G. received financial support from Syngenta Ltd. Syngenta also contributed to study design and the preparation of the manuscript, but did not have any role in data collection, data analysis, or decision to publish. R.A.B. declares no competing financial interests.

Reactive oxygen species (ROS) have been extensively studied as damaging agents associated with ageing and neurodegenerative conditions. Their role in the nervous system under non-pathological conditions has remained poorly understood. Working with the Drosophila larval locomotor network, we show that in neurons ROS act as obligate signals required for neuronal activity-dependent structural plasticity, of both pre- and postsynaptic terminals. ROS signaling is also necessary for maintaining evoked synaptic transmission at the neuromuscular junction, and for activity-regulated homeostatic adjustment of motor network output, as measured by larval crawling behavior. We identified the highly conserved Parkinson's disease-linked protein DJ-1β as a redox sensor in neurons where it regulates structural plasticity, in part via modulation of the PTEN-PI3Kinase pathway. This study provides a new conceptual framework of neuronal ROS as second messengers required for neuronal plasticity and for network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction., Competing Interests: MO, PB, MZ, AM, RW, CG, KM, RB, SS, ML No competing interests declared, (© 2018, Oswald et al.)

Global agriculture and the control of insect disease vectors have developed with a heavy reliance on insecticides. The increasing incidence of resistance, for virtually all insecticides, threatens both food supply and effective control of insect borne disease. CASPP ((5-chloro-1'-[(E)-3-(4-chlorophenyl)allyl]spiro[indoline-3,4'-piperidine]-1-yl}-(2-chloro-4-pyridyl)methanone)) compounds are a potential new class of neuroactive insecticide specifically targeting the Vesicular Acetylcholine Transporter (VAChT). Resistance to CASPP, under laboratory conditions, has been reported following either up-regulation of wildtype VAChT expression or the presence of a specific point mutation (VAChTY49N). However, the underlying mechanism of CASPP-resistance, together with the consequence to insect viability of achieving resistance, is unknown. In this study, we use electrophysiological characterisation of cholinergic release at Drosophila larval interneuron→motoneuron synapses to investigate the physiological implications of these two identified modes of CASPP resistance. We show that both VAChT up-regulation or the expression of VAChTY49N increases miniature (mini) release frequency. Mini frequency appears deterministic of CASPP activity. However, maintenance of SV release is not indicative of resistance in all cases. This is evidenced through expression of syntaxin or complexin mutants (sytx3-61/cpxSH1) that show similarly high mini release frequency but are not resistant to CASPP. The VAChTY49N mutation additionally disrupts action potential-evoked cholinergic release and fictive locomotor patterning through depletion of releasable synaptic vesicles. This observation suggests a functional trade-off for this point mutation, which is not seen when wildtype VAChT is up-regulated., Competing Interests: The authors have the following competing interests: Dr. Goodchild is employed by Syngenta Crop Protection. The insecticidal activity of the 5-CL-CASPP disclosed in this paper has been patented by Syngenta (WO2003106457, WO2005058897, WO2005061512), however the focus of this study (the molecular mechanism of action) is independent of any commercial interest, and there are no other relevant Syngenta patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. SWV and RAB declare no competing interests.

Despite availability of a diverse range of anti-epileptic drugs (AEDs), only about two-thirds of epilepsy patients respond well to drug treatment. Thus, novel targets are required to catalyse the design of next-generation AEDs. Manipulation of neuron firing-rate homoeostasis, through enhancing Pumilio (Pum) activity, has been shown to be potently anticonvulsant in Drosophila. In this study, we performed a genome-wide RNAi screen in S2R + cells, using a luciferase-based dPum activity reporter and identified 1166 genes involved in dPum regulation. Of these genes, we focused on 699 genes that, on knock-down, potentiate dPum activity/expression. Of this subgroup, 101 genes are activity-dependent based on comparison with genes previously identified as activity-dependent by RNA-sequencing. Functional cluster analysis shows these genes are enriched in pathways involved in DNA damage, regulation of cell cycle and proteasomal protein catabolism. To test for anticonvulsant activity, we utilised an RNA-interference approach in vivo. RNAi-mediated knockdown showed that 57/101 genes (61%) are sufficient to significantly reduce seizure duration in the characterized seizure mutant, para bss . We further show that chemical inhibitors of protein products of some of the genes targeted are similarly anticonvulsant. Finally, to establish whether the anticonvulsant activity of identified compounds results from increased dpum transcription, we performed a luciferase-based assay to monitor dpum promoter activity. Third instar larvae exposed to sodium fluoride, gemcitabine, metformin, bestatin, WP1066 or valproic acid all showed increased dpum promoter activity. Thus, this study validates Pum as a favourable target for AED design and, moreover, identifies a number of lead compounds capable of increasing the expression of this homeostatic regulator.

Mutations in the Golgi SNARE (SNAP [soluble NSF attachment protein] receptor) protein Membrin (encoded by the GOSR2 gene) cause progressive myoclonus epilepsy (PME). Membrin is a ubiquitous and essential protein mediating ER-to-Golgi membrane fusion. Thus, it is unclear how mutations in Membrin result in a disorder restricted to the nervous system. Here, we use a multi-layered strategy to elucidate the consequences of Membrin mutations from protein to neuron. We show that the pathogenic mutations cause partial reductions in SNARE-mediated membrane fusion. Importantly, these alterations were sufficient to profoundly impair dendritic growth in Drosophila models of GOSR2-PME. Furthermore, we show that Membrin mutations cause fragmentation of the presynaptic cytoskeleton coupled with transsynaptic instability and hyperactive neurotransmission. Our study highlights how dendritic growth is vulnerable even to subtle secretory pathway deficits, uncovers a role for Membrin in synaptic function, and provides a comprehensive explanatory basis for genotype-phenotype relationships in GOSR2-PME., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Stability of neural circuits is reliant on homeostatic mechanisms that return neuron activity towards pre-determined and physiologically appropriate levels. Without these mechanisms, changes due to synaptic plasticity, ageing and disease may push neural circuits towards instability. Whilst widely documented, understanding of how and when neurons determine an appropriate activity level, the so-called set-point, remains unknown. Genetically tractable model systems have greatly contributed to our understanding of neuronal homeostasis and continue to offer attractive models to explore these additional questions. This review focuses on the development of Drosophila motoneurons including defining an embryonic critical period during which these neurons encode their set-points to enable homeostatic regulation of activity., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Epilepsy is a significant disorder for which approximately one-third of patients do not respond to drug treatments. Next-generation drugs, which interact with novel targets, are required to provide a better clinical outcome for these individuals. To identify potential novel targets for antiepileptic drug (AED) design, we used RNA sequencing to identify changes in gene transcription in two seizure models of the fruit fly Drosophila melanogaster The first model compared gene transcription between wild type (WT) and bangsenseless 1 (para bss ), a gain-of-function mutant in the sole fly voltage-gated sodium channel (paralytic). The second model compared WT with WT fed the proconvulsant picrotoxin (PTX). We identified 743 genes (FDR≤1%) with significant altered expression levels that are common to both seizure models. Of these, 339 are consistently upregulated and 397 downregulated. We identify pumilio (pum) to be downregulated in both seizure models. Pum is a known homeostatic regulator of action potential firing in both flies and mammals, achieving control of neuronal firing through binding to, and regulating translation of, the mRNA transcripts of voltage-gated sodium channels (Na v ). We show that maintaining expression of pum in the CNS of para bss flies is potently anticonvulsive, whereas its reduction through RNAi-mediated knockdown is proconvulsive. Using a cell-based luciferase reporter screen, we screened a repurposed chemical library and identified 12 compounds sufficient to increase activity of pum Of these compounds, we focus on avobenzone, which significantly rescues seizure behaviour in para bss flies. The mode of action of avobenzone includes potentiation of pum expression and mirrors the ability of this homeostatic regulator to reduce the persistent voltage-gated Na + current (I NaP ) in an identified neuron. This study reports a novel approach to suppress seizure and highlights the mechanisms of neuronal homeostasis as potential targets for next-generation AEDs., (© 2017. Published by The Company of Biologists Ltd.)

Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca 2+ -gated Cl - channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl - ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl - conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Many animals are able to sense the Earth's geomagnetic field to enable behaviors such as migration. It is proposed that the magnitude and direction of the geomagnetic field modulates the activity of cryptochrome (CRY) by influencing photochemical radical pair intermediates within the protein. However, this proposal will remain theoretical until a CRY-dependent effect on a receptor neuron is shown to be modified by an external magnetic field (MF). It is established that blue-light (BL) photoactivation of CRY is sufficient to depolarize and activate Drosophila neurons. Here, we show that this CRY-dependent effect is significantly potentiated in the presence of an applied MF (100 mT). We use electrophysiological recordings from larval identified motoneurons, in which CRY is ectopically expressed, to show that BL-dependent depolarization of membrane potential and increased input resistance are markedly potentiated by an MF. Analysis of membrane excitability shows that these effects of MF exposure evoke increased action potential firing. Almost nothing is known about the mechanism by which a magnetically induced change in CRY activity might produce a behavioral response. We further report that specific structural changes to the protein alter the impact of the MF in ways that are strikingly similar to those from recent behavioral studies into the magnetic sense of Drosophila These observations provide the first direct experimental evidence to support the hypothesis that MF modulation of CRY activity is capable of influencing neuron activity to allow animal magnetoreception., Significance Statement: The biophysical mechanism of animal magnetoreception is still unclear. The photoreceptor protein cryptochrome has risen to prominence as a candidate magnetoreceptor molecule based on multiple reports derived from behavioral studies. However, the role of cryptochrome as a magnetoreceptor remains controversial primarily because of a lack of direct experimental evidence linking magnetic field (MF) exposure to a change in neuronal activity. Here, we show that exposure to an MF (100 mT) is sufficient to potentiate the ability of light-activated cryptochrome to increase neuronal action potential firing. Our results provide critical missing evidence to show that the activity of cryptochrome is sensitive to an external MF that is capable of modifying animal behavior., (Copyright © 2016 Giachello et al.)

Although there are now a number of antiepileptic drugs (AEDs) available, approximately one-third of epilepsy patients respond poorly to drug intervention. The reasons for this are complex, but are probably reflective of the increasing number of identified mutations that predispose individuals to this disease. Thus, there is a clear requirement for the development of novel treatments to address this unmet clinical need. The existence of gene mutations that mimic a seizure-like behaviour in the fruit fly, Drosophila melanogaster, offers the possibility to exploit the powerful genetics of this insect to identify novel cellular targets to facilitate design of more effective AEDs. In this study we use neuronal expression of GCaMP, a potent calcium reporter, to image neuronal activity using a non-invasive and rapid method. Expression in motoneurons in the isolated CNS of third instar larvae shows waves of calcium-activity that pass between segments of the ventral nerve cord. Time between calcium peaks, in the same neurons, between adjacent segments usually show a temporal separation of greater than 200 ms. Exposure to proconvulsants (picrotoxin or 4-aminopyridine) reduces separation to below 200 ms showing increased synchrony of activity across adjacent segments. Increased synchrony, characteristic of epilepsy, is similarly observed in genetic seizure mutants: bangsenseless1 (bss1) and paralyticK1270T (paraK1270T). Exposure of bss1 to clinically-used antiepileptic drugs (phenytoin or gabapentin) significantly reduces synchrony. In this study we use the measure of synchronicity to evaluate the effectiveness of known and novel anticonvulsive compounds (antipain, isethionate, etopiside rapamycin and dipyramidole) to reduce seizure-like CNS activity. We further show that such compounds also reduce the Drosophila voltage-gated persistent Na+ current (INaP) in an identified motoneuron (aCC). Our combined assays provide a rapid and reliable method to screen unknown compounds for potential to function as anticonvulsants.

Experimental evidence shows that neurotransmitter release, from presynaptic terminals, can be regulated by altering transmitter load per synaptic vesicle (SV) and/or through change in the probability of vesicle release. The vesicular acetylcholine transporter (VAChT) loads acetylcholine into SVs at cholinergic synapses. We investigated how the VAChT affects SV content and release frequency at central synapses in Drosophila melanogaster by using an insecticidal compound, 5Cl-CASPP, to block VAChT and by transgenic overexpression of VAChT in cholinergic interneurons. Decreasing VAChT activity produces a decrease in spontaneous SV release with no change to quantal size and no decrease in the number of vesicles at the active zone. This suggests that many vesicles are lacking in neurotransmitter. Overexpression of VAChT leads to increased frequency of SV release, but again with no change in quantal size or vesicle number. This indicates that loading of central cholinergic SVs obeys the "set-point" model, rather than the "steady-state" model that better describes loading at the vertebrate neuromuscular junction. However, we show that expression of a VAChT polymorphism lacking one glutamine residue in a COOH-terminal polyQ domain leads to increased spontaneous SV release and increased quantal size. This effect spotlights the poly-glutamine domain as potentially being important for sensing the level of neurotransmitter in cholinergic SVs., (Copyright © 2016 the American Physiological Society.)

Maturation of neural circuits requires activity-dependent processes that underpin the emergence of appropriate behavior in the adult. It has been proposed that disruption of these events, during specific critical periods when they exert maximal influence, may lead to neurodevelopmental diseases, including epilepsy. However, complexity of neurocircuitry, coupled with the lack of information on network formation in mammals, makes it difficult to directly investigate this hypothesis. Alternative models, including the fruit fly Drosophila melanogaster, show remarkable similarities between experimental seizure-like activity and clinical phenotypes. In particular, a group of flies, termed bang-sensitive (bs) mutants have been extensively used to investigate the pathophysiological mechanisms underlying seizure. Seizure phenotype can be measured in larval stages using an electroshock assay, and this behavior in bs mutants is dramatically reduced following ingestion of typical anti-epileptic drugs (AEDs;). In this study we describe a critical period of embryonic development in Drosophila during which manipulation of neural activity is sufficient to significantly influence seizure behavior at postembryonic stages. We show that inhibition of elevated activity, characteristic of bs seizure models, during the critical period is sufficient to suppress seizure. By contrast, increasing neuronal excitation during the same period in wild-type (WT) is sufficient to permanently induce a seizure behavior. Further, we show that induction of seizure in WT correlates with functional alteration of motoneuron inputs that is a characteristic of bs mutants. Induction of seizure is rescued by prior administration of AEDs, opening a new perspective for early drug intervention in the treatment of genetic epilepsy., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Studying ion channel currents generated distally from the recording site is difficult because of artifacts caused by poor space clamp and membrane filtering. A computational model can quantify artifact parameters for correction by simulating the currents only if their exact anatomical location is known. We propose that the same artifacts that confound current recordings can help pinpoint the source of those currents by providing a signature of the neuron's morphology. This method can improve the recording quality of currents initiated at the spike initiation zone (SIZ) that are often distal to the soma in invertebrate neurons. Drosophila being a valuable tool for characterizing ion currents, we estimated the SIZ location and quantified artifacts in an identified motoneuron, aCC/MN1-Ib, by constructing a novel multicompartmental model. Initial simulation of the measured biophysical channel properties in an isopotential Hodgkin-Huxley type neuron model partially replicated firing characteristics. Adding a second distal compartment, which contained spike-generating Na+ and K+ currents, was sufficient to simulate aCC's in vivo activity signature. Matching this signature using a reconstructed morphology predicted that the SIZ is on aCC's primary axon, 70 μm after the most distal dendritic branching point. From SIZ to soma, we observed and quantified selective morphological filtering of fast activating currents. Non-inactivating K+ currents are filtered ∼3 times less and despite their large magnitude at the soma they could be as distal as Na+ currents. The peak of transient component (NaT) of the voltage-activated Na+ current is also filtered more than the magnitude of slower persistent component (NaP), which can contribute to seizures. The corrected NaP/NaT ratio explains the previously observed discrepancy when the same channel is expressed in different cells. In summary, we used an in vivo signature to estimate ion channel location and recording artifacts, which can be applied to other neurons.

Seizure can result from increased voltage-gated persistent sodium current expression. Although many clinically-approved antiepileptic drugs target voltage-gated persistent sodium current, none exclusively repress this current without also adversely affecting the transient voltage-gated sodium current. Achieving a more selective block has significant potential for the treatment of epilepsy. Recent studies show that voltage-gated persistent sodium current amplitude is regulated by alternative splicing offering the possibility of a novel route for seizure control. In this study we identify 291 splicing regulators that, on knockdown, alter splicing of the Drosophila voltage-gated sodium channel to favour inclusion of exon K, rather than the mutually exclusive exon L. This change is associated with both a significant reduction in voltage-gated persistent sodium current, without change to transient voltage-gated sodium current, and to rescue of seizure in this model insect. RNA interference mediated knock-down, in two different seizure mutants, shows that 95 of these regulators are sufficient to significantly reduce seizure duration. Moreover, most suppress seizure activity in both mutants, indicative that they are part of well conserved pathways and likely, therefore, to be optimal candidates to take forward to mammalian studies. We provide proof-of-principle for such studies by showing that inhibition of a selection of regulators, using small molecule inhibitors, is similarly effective to reduce seizure. Splicing of the Drosophila sodium channel shows many similarities to its mammalian counterparts, including altering the amplitude of voltage-gated persistent sodium current. Our study provides the impetus to investigate whether manipulation of splicing of mammalian voltage-gated sodium channels may be exploitable to provide effective seizure control., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.)

The voltage-gated sodium channel (Nav) plays a key role in regulation of neuronal excitability. Aberrant regulation of Nav expression and/or function can result in an imbalance in neuronal activity which can progress to epilepsy. Regulation of Nav activity is achieved by coordination of a multitude of mechanisms including RNA alternative splicing and translational repression. Understanding of these regulatory mechanisms is complicated by extensive genetic redundancy: the mammalian genome encodes ten Navs. By contrast, the genome of the fruitfly, Drosophila melanogaster, contains just one Nav homologue, encoded by paralytic (DmNa v ). Analysis of splicing in DmNa v shows variants exhibit distinct gating properties including varying magnitudes of persistent sodium current (INaP). Splicing by Pasilla, an identified RNA splicing factor, alters INaP magnitude as part of an activity-dependent mechanism. Enhanced INaP promotes membrane hyperexcitability that is associated with seizure-like behaviour in Drosophila. Nova-2, a mammalian Pasilla homologue, has also been linked to splicing of Navs and, moreover, mouse gene knockouts display seizure-like behaviour.Expression level of Navs is also regulated through a mechanism of translational repression in both flies and mammals. The translational repressor Pumilio (Pum) can bind to Na v transcripts and repress the normal process of translation, thus regulating sodium current (INa) density in neurons. Pum2-deficient mice exhibit spontaneous EEG abnormalities. Taken together, aberrant regulation of Nav function and/or expression is often epileptogenic. As such, a better understanding of regulation of membrane excitability through RNA alternative splicing and translational repression of Navs should provide new leads to treat epilepsy.

This narrative review is intended to introduce clinicians treating epilepsy and researchers familiar with mammalian models of epilepsy to experimentally tractable, non-mammalian research models used in epilepsy research, ranging from unicellular eukaryotes to more complex multicellular organisms. The review focuses on four model organisms: the social amoeba Dictyostelium discoideum, the roundworm Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the zebrafish Danio rerio. We consider recent discoveries made with each model organism and discuss the importance of these advances for the understanding and treatment of epilepsy in humans. The relative ease with which mutations in genes of interest can be produced and studied quickly and cheaply in these organisms, together with their anatomical and physiological simplicity in comparison to mammalian species, are major advantages when researchers are trying to unravel complex disease mechanisms. The short generation times of most of these model organisms also mean that they lend themselves particularly conveniently to the investigation of drug effects or epileptogenic processes across the lifecourse., (Copyright © 2014 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

The mechanisms that facilitate animal magnetoreception have both fascinated and confounded scientists for decades, and its precise biophysical origin remains unclear. Among the proposed primary magnetic sensors is the flavoprotein, cryptochrome, which is thought to provide geomagnetic information via a quantum effect in a light-initiated radical pair reaction. Despite recent advances in the radical pair model of magnetoreception from theoretical, molecular and animal behaviour studies, very little is known of a possible signal transduction mechanism. We report a substantial effect of magnetic field exposure on seizure response in Drosophila larvae. The effect is dependent on cryptochrome, the presence and wavelength of light and is blocked by prior ingestion of typical antiepileptic drugs. These data are consistent with a magnetically-sensitive, photochemical radical pair reaction in cryptochrome that alters levels of neuronal excitation, and represent a vital step forward in our understanding of the signal transduction mechanism involved in animal magnetoreception.

Miniature neurotransmission is the transsynaptic process where single synaptic vesicles spontaneously released from presynaptic neurons induce miniature postsynaptic potentials. Since their discovery over 60 years ago, miniature events have been found at every chemical synapse studied. However, the in vivo necessity for these small-amplitude events has remained enigmatic. Here, we show that miniature neurotransmission is required for the normal structural maturation of Drosophila glutamatergic synapses in a developmental role that is not shared by evoked neurotransmission. Conversely, we find that increasing miniature events is sufficient to induce synaptic terminal growth. We show that miniature neurotransmission acts locally at terminals to regulate synapse maturation via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Our results establish that miniature neurotransmission, a universal but often-overlooked feature of synapses, has unique and essential functions in vivo., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Our previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. In this study, we use the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K(+) channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. We used body wall muscle as a test tissue because in conditions of low Ca(2+), the fast K(+) current is carried solely by Sh channels (unlike neurons in which a second fast K(+) current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression.

RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar(5G1) null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar(5G1) mutant phenotypes are ameliorated by feeding GABA modulators. We demonstrate that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar(5G1) null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability.

The human brain comprises approximately 100 billion neurons that express a diverse, and often subtype-specific, set of neurotransmitters and voltage-gated ion channels. Given this enormous complexity, a fundamental question is how is this achieved? The acquisition of neurotransmitter phenotype was viewed as being set by developmental programs 'hard wired' into the genome. By contrast, the expression of neuron-specific ion channels was considered to be highly dynamic (i.e., 'soft wired') and shaped largely by activity-dependent mechanisms. Recent evidence blurs this distinction by showing that neurotransmitter phenotype can be altered by activity and that neuron type-specific ion channel expression can be set, and perhaps limited by, developmental programs. Better understanding of these early regulatory mechanisms may offer new avenues to avert the behavioral changes that are characteristic of many mental illnesses., (Copyright © 2013. Published by Elsevier Ltd.)

Innexins are one of two gene families that have evolved to permit neighbouring cells in multicellular systems to communicate directly. Innexins are found in prechordates and persist in small numbers in chordates as divergent sequences termed pannexins. Connexins are functionally analogous proteins exclusive to chordates. Members of these two families of proteins form intercellular channels, assemblies of which constitute gap junctions. Each intercellular channel is a composite of two hemichannels, one from each of two apposed cells. Hemichannels dock in the extracellular space to form a complete channel with a central aqueous pore that regulates the cell-cell exchange of ions and small signalling molecules. Hemichannels can also act independently by releasing paracrine signalling molecules. optic ganglion reduced (ogre) is a member of the Drosophila innexin family, originally identified as a gene essential for postembryonic neurogenesis. Here we demonstrate, by heterologous expression in paired Xenopus oocytes, that Ogre alone does not form homotypic gap-junction channels; however, co-expression of Ogre with Innexin2 (Inx2) induces formation of functional channels with properties distinct from Inx2 homotypic channels. In the Drosophila larval central nervous system, we find that Inx2 partially colocalises with Ogre in proliferative neuroepithelia and in glial cells. Downregulation of either ogre or inx2 selectively in glia, by targeted expression of RNA interference transgenes, leads to a significant reduction in the size of the larval nervous system and behavioural defects in surviving adults. We conclude that these innexins are crucially required in glial cells for normal postembryonic development of the central nervous system.

The ability to regulate intrinsic membrane excitability, to maintain consistency of action potential firing, is critical for stable neural circuit activity. Without such mechanisms, Hebbian-based synaptic plasticity could push circuits toward activity saturation or, alternatively, quiescence. Although now well documented, the underlying molecular components of these homeostatic mechanisms remain poorly understood. Recent work in the fruit fly, Drosophila melanogaster, has identified Pumilio (Pum), a translational repressor, as an essential component of one such mechanism. In response to changing synaptic excitation, Pum regulates the translation of the voltage-gated sodium conductance, leading to a concomitant adjustment in action potential firing. Although similar homeostatic mechanisms are operational in mammalian neurons, it is unknown whether Pum is similarly involved. In this study, we report that Pum2 is indeed central to the homeostatic mechanism regulating membrane excitability in rat visual cortical pyramidal neurons. Using RNA interference, we observed that loss of Pum2 leads to increased sodium current (I(Na)) and action potential firing, mimicking the response by these neurons to being deprived of synaptic depolarization. In contrast, increased synaptic depolarization results in increased Pum2 expression and subsequent reduction in INa and membrane excitability. We further show that Pum2 is able to directly bind the predominant voltage-gated sodium channel transcript (NaV1.6) expressed in these neurons and, through doing so, regulates translation of this key determinant of membrane excitability. Together, our results show that Pum2 forms part of a homeostatic mechanism that matches membrane excitability to synaptic depolarization in mammalian neurons.

Neuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K(+) currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K(+) current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Activity of voltage-gated Na channels (Na(v)) is modified by alternative splicing. However, whether altered splicing of human Na(v)s contributes to epilepsy remains to be conclusively shown. We show here that altered splicing of the Drosophila Na(v) (paralytic, DmNa(v)) contributes to seizure-like behavior in identified seizure mutants. We focus attention on a pair of mutually exclusive alternate exons (termed K and L), which form part of the voltage sensor (S4) in domain III of the expressed channel. The presence of exon L results in a large, non-inactivating, persistent I(Nap). Many forms of human epilepsy are associated with an increase in this current. In wild-type (WT) Drosophila larvae, ∼70-80% of DmNa(v) transcripts contain exon L, and the remainder contain exon K. Splicing of DmNa(v) to include exon L is increased to ∼100% in both the slamdance and easily-shocked seizure mutants. This change to splicing is prevented by reducing synaptic activity levels through exposure to the antiepileptic phenytoin or the inhibitory transmitter GABA. Conversely, enhancing synaptic activity in WT, by feeding of picrotoxin is sufficient to increase I(Nap) and promote seizure through increased inclusion of exon L to 100%. We also show that the underlying activity-dependent mechanism requires the presence of Pasilla, an RNA-binding protein. Finally, we use computational modeling to show that increasing I(Nap) is sufficient to potentiate membrane excitability consistent with a seizure phenotype. Thus, increased synaptic excitation favors inclusion of exon L, which, in turn, further increases neuronal excitability. Thus, at least in Drosophila, this self-reinforcing cycle may promote the incidence of seizure.

The fruit fly Drosophila melanogaster has been instrumental in expanding our understanding of early aspects of neural development. The use of this model system has greatly added to our knowledge of neural cell-fate determination, axon guidance, and synapse formation. It has also become possible to access and make electrophysiological recordings directly from neurons in situ in an intact central nervous system (CNS), which has facilitated studies of the development and regulation of neuronal signaling. It is possible to obtain electrophysiological recordings from all stages of Drosophila. Exposure of the intact Drosophila CNS is a prerequisite for such electrophysiological recordings. The dissection procedure described here can be applied to both late-stage embryos (stage 16 onward) and larvae. Because of their size, third-instar larvae are more difficult to flatten using this method and, if recording from this stage, the reader might consider using insect pins for the dissection or isolating the CNS using an alternative method. The dissection should take <10 min if all preparation work has been completed in advance. Owing to the short life span of the dissected larva, it is not recommended that the procedure be stopped or the preparation stored for later use.

The fruit fly Drosophila melanogaster has been instrumental in expanding our understanding of early aspects of neural development. The use of this model system has greatly added to our knowledge of neural cell-fate determination, axon guidance, and synapse formation. It has also become possible to access and make electrophysiological recordings directly from neurons in situ in an intact central nervous system (CNS), which has facilitated studies of the development and regulation of neuronal signaling. This protocol describes a procedure for revealing larval motor neurons and applying whole-cell patch recording techniques to these cells. The useful lifetime of first-instar larval preparations is ∼30 min, and that of third-instar CNS preparations is up to 1 h. It is therefore recommended that fresh preparations are used and that no breaks are taken during the procedure, although there may be time to pull and polish a patch pipette.