Ex vivo Whole-cell Recordings in Adult Drosophila Brain

Roemmich, Alexa J.; Schutte, Soleil S.; O’Dowd, Diane K.

doi:10.21769/bioprotoc.2467

Cited by 6 publications

(4 citation statements)

References 23 publications

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“…Though the site of seizure generation in flies is unknown, LNs in the fly antennal lobes are a readily accessible population of GABAergic inhibitory neurons in the important and well characterized olfactory circuit within the Drosophila brain ( Seki et al, 2010 ). To observe neuronal firing and sodium channel activity in these neurons, the whole brain was dissected out of adult flies ( Roemmich et al, 2018 ), dorsal lateral local neurons were identified by their location and morphology, and whole-cell electrophysiological recordings were performed.…”

Section: Resultsmentioning

confidence: 99%

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Seizure Phenotype and Underlying Cellular Defects inDrosophilaKnock-In Models of DS (R1648C) and GEFS+ (R1648H)SCN1AEpilepsy

Roemmich

Lukácsovich

et al. 2021

eNeuro

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Seizure phenotype and underlying cellular defects in Drosophila knock-in models of DS (R1648C) and GEFS+ (R1648H) SCN1A epilepsy ABSTRACT Mutations in the voltage-gated sodium channel gene SCN1A are associated with human epilepsy disorders, but how most of these mutations alter channel properties and result in seizures is unknown. This study focuses on two different mutations occurring at one position within SCN1A. R1648C is associated with the severe disorder Dravet Syndrome, and R1648H, with the milder disorder GEFS+. To explore how these different mutations contribute to distinct seizure disorders, Drosophila lines with the R-C or R-H mutation, or R-R control substitution in the fly sodium channel gene para were generated by CRISPR-Cas9 gene editing. The R-C and R-H mutations are homozygous lethal. Animals heterozygous for R-C or R-H mutations displayed reduced lifespans and spontaneous and temperature-induced seizures not observed in R-R controls.Electrophysiological recordings from adult GABAergic neurons in R-C and R-H mutants revealed appearance of sustained neuronal depolarizations and altered firing frequency that were exacerbated at elevated temperature. The only significant change observed in underlying sodium currents in both R-C and R-H mutants was a hyperpolarized deactivation threshold at room and elevated temperature compared to R-R controls.Since this change is constitutive it is likely to interact with heat-induced changes in other cellular properties to result in the heat-induced increase in sustained depolarizations and seizure activity. Further, the similarity of the behavioral and cellular phenotypes in the R-C and R-H fly lines, suggests that disease symptoms of different severity 3 associated with these mutations in humans could be due in large part to differences in genetic background. SIGNIFICANCE STATEMENT: Knock-in Drosophila lines were generated by CRISPR-Cas9 gene editing to explore how the R1648C and R1648H mutations in the SCN1A sodium channel gene contribute to distinct epilepsy disorders, Dravet syndrome and GEFS+ respectively. Drosophila heterozygous for the R-C or R-H mutation displayed spontaneous seizures exacerbated at high temperature. GABAergic neurons in both mutants exhibited sustained depolarizations, interrupting neuronal firing, that increased in incidence at elevated temperature. A hyperpolarized deactivation threshold in R-C and R-H sodium currents was constitutive implicating interaction with other heatsensitive processes to alter firing properties. The similarity of the behavioral and cellular phenotypes in the R-C and R-H fly lines suggests that disorders of different severity in humans could be due in large part to differences in genetic background.

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Section: Resultsmentioning

confidence: 99%

“…al., 2010). To observe neuronal firing and sodium channel activity in these neurons, the whole brain was dissected out of adult flies (Roemmich et. al., 2018), dorsal lateral local neurons were identified by their location and morphology, and whole-cell electrophysiological recordings were performed.…”

Section: R1648h Inhibitory Neuronsmentioning

confidence: 99%

Seizure Phenotype and Underlying Cellular Defects inDrosophilaKnock-In Models of DS (R1648C) and GEFS+ (R1648H)SCN1AEpilepsy

Roemmich

Lukácsovich

et al. 2021

eNeuro

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“…While we have yet to observe positive results in any epilepsy candidate genes, these methods can provide a strong tool for studying genes involved in synaptic function coding in epilepsy. Combining these electrophysiological methods with the LPF recording technique in the CNS of Drosophila ( Iyengar and Wu, 2021 ) can develop an instrumental platform for investigating the cellular and synaptic mechanisms underlying neural function or dysfunction ( Roemmich et al, 2018 ), with excellent accessibility for studying epilepsy’s pathological and pharmacological aspects ( Sheeba et al, 2008 ).…”

Section: Discussionmentioning

confidence: 99%

Efficient strategies based on behavioral and electrophysiological methods for epilepsy-related gene screening in the Drosophila model

Liu

et al. 2023

Front. Mol. Neurosci.

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IntroductionWith the advent of trio-based whole-exome sequencing, the identification of epilepsy candidate genes has become easier, resulting in a large number of potential genes that need to be validated in a whole-organism context. However, conducting animal experiments systematically and efficiently remains a challenge due to their laborious and time-consuming nature. This study aims to develop optimized strategies for validating epilepsy candidate genes using the Drosophila model.MethodsThis study incorporate behavior, morphology, and electrophysiology for genetic manipulation and phenotypic examination. We utilized the Gal4/UAS system in combination with RNAi techniques to generate loss-of-function models. We performed a range of behavioral tests, including two previously unreported seizure phenotypes, to evaluate the seizure behavior of mutant and wild-type flies. We used Gal4/UAS-mGFP flies to observe the morphological alterations in the brain under a confocal microscope. We also implemented patch-clamp recordings, including a novel electrophysiological method for studying synapse function and improved methods for recording action potential currents and spontaneous EPSCs on targeted neurons.ResultsWe applied different techniques or methods mentioned above to investigate four epilepsy-associated genes, namely Tango14, Klp3A, Cac, and Sbf, based on their genotype-phenotype correlation. Our findings showcase the feasibility and efficiency of our screening system for confirming epilepsy candidate genes in the Drosophila model.DiscussionThis efficient screening system holds the potential to significantly accelerate and optimize the process of identifying epilepsy candidate genes, particularly in conjunction with trio-based whole-exome sequencing.

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“…Flies have also become convenient models to perform neuronal cell recording in the adult brain (ex vivo) that can be studies in the context of genetic and/or pharmacological manipulations (Gu and O'dowd, 2007;Roemmich et al, 2018). As an example, electrophysiological studies carried out in adult flies that were genetically modified to mimic DS were pioneers in showing the link between pathological missense mutations and disturbances of sodium ion current activity at the receptor level (Sun et al, 2012;Schutte et al, 2014).…”

Section: From Gene Mutation To Brain Network Perturbation (Temporal Mmentioning

confidence: 99%

Functional Genomics of Epilepsy and Associated Neurodevelopmental Disorders Using Simple Animal Models: From Genes, Molecules to Brain Networks

Rosch

Burrows

Jones

et al. 2019

Front. Cell. Neurosci.

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The genetic diagnosis of patients with seizure disorders has been improved significantly by the development of affordable next-generation sequencing technologies. Indeed, in the last 20 years, dozens of causative genes and thousands of associated variants have been described and, for many patients, are now considered responsible for their disease. However, the functional consequences of these mutations are often not studied in vivo, despite such studies being central to understanding pathogenic mechanisms and identifying novel therapeutic avenues. One main roadblock to functionally characterizing pathogenic mutations is generating and characterizing in vivo mammalian models carrying clinically relevant variants in specific genes identified in patients. Although the emergence of new mutagenesis techniques facilitates the production of rodent mutants, the fact that early development occurs internally hampers the investigation of gene function during neurodevelopment. In this context, functional genomics studies using simple animal models such as flies or fish are advantageous since they open a dynamic window of investigation throughout embryonic development. In this review, we will summarize how the use of simple animal models can fill the gap between genetic diagnosis and functional and phenotypic correlates of gene function in vivo. In particular, we will discuss how these simple animals offer the possibility to study gene function at multiple scales, from molecular function (i.e., ion channel activity), to cellular circuit and brain network dynamics. As a result, simple model systems offer alternative avenues of investigation to model aspects of the disease phenotype not currently possible in rodents, which can help to unravel the pathogenic substratum in vivo.

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Ex vivo Whole-cell Recordings in Adult Drosophila Brain

Cited by 6 publications

References 23 publications

Seizure Phenotype and Underlying Cellular Defects inDrosophilaKnock-In Models of DS (R1648C) and GEFS+ (R1648H)SCN1AEpilepsy

Seizure Phenotype and Underlying Cellular Defects inDrosophilaKnock-In Models of DS (R1648C) and GEFS+ (R1648H)SCN1AEpilepsy

Efficient strategies based on behavioral and electrophysiological methods for epilepsy-related gene screening in the Drosophila model

Functional Genomics of Epilepsy and Associated Neurodevelopmental Disorders Using Simple Animal Models: From Genes, Molecules to Brain Networks

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