bang-sensitive assay for the study of seizure-like behavior in drosophila

Monitoreo de eventos epileptiformes en Drosophila a través de imágenes de calcio

Epilepsy, a complex chronic neurological disorder characterized by the recurrence of spontaneous and unprovoked seizures and aberrant neuronal network activity, has affected over 70 million individuals worldwide, making it one of the most common neurological diseases1 and leading to the heavy burdens of families and society. In consideration of the impact of epilepsy, many studies have been conducted to identify the etiology of seizures, of which genetics has been approved as a primary cause of many types of epilepsies or epileptic syndromes2. For the past decades, advances in genomic technologies have led to a rapid increase in the discovery of novel epilepsy-associated genes, which play a crucial role in seizure occurrence, including ion channels and non-ion channel genes3,4. However, the underlying mechanisms and functional analysis between the genes and epileptic phenotypes are incompletely understood. Identifying epilepsy-associated genes and mechanisms offers the possibility to the management of patients efficiently5,6.
Cytosolic calcium signals are pivotal elements in neuronal activity and synaptic transmission. Calcium imaging, including brain slices7, in vivo8,9, and ex vivo10, has been utilized to monitor neuronal activity11 as a marker for neuronal excitability since the 1970s12,13. Recent advancements in imaging technology, in combination with the genetically encoded calcium indicators (GECIs), such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels14,15,16, which has a high level of spatiotemporal precision. Changes in calcium concentration and transients were observed in action potentials and synaptic transmission, respectively14, indicating the alteration of intracellular calcium levels exhibits a strict correlation with the electrical excitability of neurons17,18. Calcium imaging has also been applied as a developmental seizure model9 and performed in Drosophila for screening anticonvulsive compounds19.
Drosophila melanogaster has been emerging as a powerful model organism in scientific research, such as epilepsy, for its sophisticated molecular genetics and behavioral assays20,21,22. Moreover, the advanced genetic tools in Drosophila have contributed to the expression of genetically encoded calcium indicator GCaMP6. For instance, the Gal4 and UAS-based binary transcriptional systems enable specific expression of the GCaMP6 in a spatially and temporally controlled manner. Since Drosophila is a tiny organism, in vivo calcium imaging requires proficient operation skills to perform a surgical intervention, in which only a small part of the dorsal of the brain was exposed through a small window14,23. At the same time, ex vivo calcium imaging in the intact brain of Drosophila can be used to monitor the regions of interest (ROIs) of the whole brain.
In this study, we present ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. CACNA1A is a well-known epilepsy gene, cac belongs to Cav2 channel, which is a homolog to CACNA1A. We began by dissecting the brains of cac knockdown flies tub-Gal4&#62;GCaMP6m/cac-RNAi and imaging them using a confocal microscope with xyt scanning mode. We then analyzed the changes in calcium signals of ROIs by calculating indicators that quantify spontaneous seizure-like events, such as %&#916;F/F value and calcium events of GCaMP6 fluorescence. Additionally, we performed mechanical stimulus by vortex machine to induce seizure behavior tests on cac-knockdown flies as well to validate the results of calcium imaging. Overall, this protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Autor destacado: Perspectivas sobre las técnicas y los hallazgos de los avances recientes en la investigación de la epilepsia 

Ex Vivo Imágenes de calcio para el modelo de epilepsia de Drosophila

Watch this Scientific Journal Video about Bang-Sensitive Assay for the Study of Seizure-Like Behavior in Drosophila at JoVE.com

Bang-Sensitive Assay for the Study of Seizure-Like Behavior in Drosophila  

Ensayo sensible a explosiones para el estudio del comportamiento similar a las convulsiones en Drosophila

Para empezar, recolecta las moscas vírgenes de la línea híbrida tub-Gal4 y las moscas macho de la línea UAS-cac-RNAi. Transfiera las moscas vírgenes y macho al mismo frasco para cosechar la descendencia. Después de tres a cinco días de oclusión, use un cepillo para recolectar las crías y las moscas tub-GL4 UAS-cac-RNA.Un día antes de la prueba, transfiera las moscas a viales nuevos y limpios con comida. A continuación, coloque cuidadosamente de cuatro a seis moscas anestesiadas con dióxido de carbono en viales frescos individuales. Coloque una cámara en un trípode frente a una pizarra.Ajuste manualmente el enfoque de la cámara con un vial vacío. Ahora, haga un vórtice de los viales con las moscas en la configuración más alta durante 10 segundos. Coloque inmediatamente el frasco en la pizarra y observe a las moscas para detectar cualquier comportamiento similar a una convulsión.Mida el tiempo de recuperación como el tiempo requerido para que las moscas recuperen la capacidad de mantenerse erguidas. Las moscas de derribo de cac mostraron tasas significativamente más altas de comportamiento similar a las convulsiones que las moscas de tipo salvaje. El porcentaje de recuperación de las moscas derribadas en un segundo fue significativamente menor que el de las moscas de tipo salvaje.

bang-sensitive-assay-for-study-seizure-like-behavior

Research

JoVE Journal

Neuroscience

Bang-Sensitive Assay for the Study of Seizure-Like Behavior in Drosophila

246 vistas. Para empezar, recolecta las moscas vírgenes de la línea híbrida tub-Gal4 y las moscas macho de la línea UAS-cac-RNAi. Transfiera las moscas vírgenes y macho al mismo frasco para cosechar la descendencia. Después de tres a cinco días de oclusión, use un cepillo para recolectar las crías y las moscas tub-GL4 UAS-cac-RNA.Un día antes de la prueba, transfiera las moscas a viales nuevos y limpios con comida. A continuación, coloque cuidadosamente de cuatro a seis moscas anestesia...

Video: Bang-Sensitive Assay for the Study of Seizure-Like Behavior in Drosophila  

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...