calcium imaging of drosophila ex vivo brain for epileptiform analysis

Monitoramento de eventos epileptiformes em Drosophila por meio de imagens de cálcio

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 em destaque: insights sobre as técnicas e descobertas de avanços recentes na pesquisa da epilepsia 

Ex Vivo Imagem de cálcio para Drosophila Modelo de Epilepsia

Recently, many epilepsy candidate gene have been found in gene tests and then readily by animal experiments. We deliver a set of techniques to study the epilepsy-related neural activity, including whole cell recording, evoked EPSP recording and the new one that we will introduce here, ex vivo calcium imaging. Since the integrity of the brain and its neural networks cannot be fully replicated in cell culture or brain slices, the main advantage of the current experiment is to obtain intact brain tissue while protecting neural networks from damage.We have established an ex vivo calcium imaging technique along with the band-sensitive seizure-like behavior assay for efficiently screening the epilepsy-associated genes and exploring the underlying mechanisms of epilepsy at the cellular level. We implemented isolated, intact Drosophila brain tissues for calcium imaging, which can avoid their complex surgical techniques and preserve the integrity of neural networks. And the ex vivo approach can also yield the superior signal to noise ratio when compared with the in vivo imaging techniques.

Watch this Scientific Journal Video about Calcium Imaging of Drosophila Ex Vivo Brain for Epileptiform Analysis at JoVE.com

Calcium Imaging of Drosophila Ex Vivo Brain for Epileptiform Analysis  

Imagem de cálcio do cérebro de Drosophila ex vivo para análise epileptiforme

Para começar, perfundir a solução externa com soro fisiológico oxigenado por cinco minutos. Com uma pipeta, transfira 10 microlitros da solução de dissecção para uma placa de Petri. Agora, use agulhas de seringa e um microscópio para dissecar cuidadosamente os cérebros das moscas selvagens estabelecidas anestesiadas e nocauteadas.Com uma pipeta, transfira o cérebro preparado para uma placa de gravação com cinco mililitros de solução externa. Imobilize os cérebros com um suporte afiado em C. Em seguida, capture a imagem confocal de cada cérebro em 20x.Use o modo de varredura XYT e identifique os neurônios do corpo do cogumelo em uma amplificação digital adicional de 4,5 vezes. Ajuste a excitação do laser para 488 nanômetros com potência de laser de 16 microwatts para adquirir toda a emissão de GCaMP6m do cérebro. Em seguida, defina os parâmetros de digitalização para velocidade de 1400 com um tamanho de pixel de 256 por 256 pixels.Defina a taxa de aquisição para 5,3 hertz e recorde por três minutos. Analisar a fluorescência de cinco a oito regiões de interesse. Determinar manualmente o corpo celular dos neurônios do corpo do cogumelo como a região de interesse.Use a imagem J para marcar os neurônios identificados e medir suas intensidades de fluorescência. Analise os dados de fluorescência do GCaMP6m conforme mostrado. Defina a fluorescência intracelular aumentando entre dois e 2,5 desvios padrão como pequenas espículas e aquelas aumentando mais de 2,5 desvios padrão como grandes espículas.Sinais de cálcio foram observados no corpo do cogumelo das moscas. As moscas knockdown do cac mostraram mais picos grandes do que pequenos.

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Research

JoVE Journal

Neuroscience

Calcium Imaging of Drosophila Ex Vivo Brain for Epileptiform Analysis

184 visualizações.  the Second Affiliated Hospital, Guangzhou Medical University. Para começar, perfundir a solução externa com soro fisiológico oxigenado por cinco minutos. Com uma pipeta, transfira 10 microlitros da solução de dissecção para uma placa de Petri. Agora, use agulhas de seringa e um microscópio para dissecar cuidadosamente os cérebros das moscas selvagens estabelecidas anestesiadas e nocauteadas.Com uma pipeta, transfira o cérebro preparado para uma placa de gravação com cinco mililitros de solução externa. Imobilize os cérebros com um...

Vídeo: Calcium Imaging of Drosophila Ex Vivo Brain for Epileptiform Analysis  

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 ...

Neurociência

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

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

To begin, collect the virgin flies of the tub-Gal4 hybrid line and the male flies of the UAS-cac-RNAi line. Transfer the virgin and male flies into the same vial to harvest the offspring. After three to five days of occlusion, use a brush to collect the offspring and the tub-GL4 UAS-cac-RNA flies.A day before testing, transfer the flies to new clean vials with food. Next, carefully place four to six carbon dioxide anesthetized flies into individual fresh vials. Place a camera on a tripod in front of a whiteboard.Manually adjust the camera's focus with an empty vial. Now, vortex the vials with the flies at the highest setting for 10 seconds. Immediately place the vial on the whiteboard and observe the flies for any seizure-like behavior.Measure the recovery time as the time required for the flies to regain the ability to stand upright. The cac knockdown flies showed significantly higher rates of seizure-like behavior than the wild type flies. The recovery percentage of knockdown flies within one second was significantly lower than the wild type flies.

Ensaio Sensível a Bang para o Estudo do Comportamento Semelhante a Convulsões em Drosophila

To begin, perfuse the external solution with oxygenated saline for five minutes. With a pipette, transfer 10 microliters of the dissection solution to a Petri dish. Now, use syringe needles and a microscope to carefully dissect the brains of the anesthetized established wild type and knockout flies.With a pipette, transfer the prepared brain into a recording dish with five milliliters of external solution. Immobilize the brains with a C sharp holder. Next, capture the confocal image of each brain at 20x.Use the XYT scanning mode and identify the mushroom body neurons at an additional 4.5 times digital amplification. Set the laser excitation to 488 nanometers with 16 micro watt laser power to acquire the whole brain GCaMP6m emission. Then set the scanning parameters to 1400 speed with a pixel size of 256 by 256 pixels.Set the acquisition rate to 5.3 hertz and record for three minutes. Analyze the fluorescence of five to eight regions of interest. Manually determine the cell body of mushroom body neurons as the region of interest.Use image J to label the identified neurons and measure their fluorescence intensities. Analyze the fluorescence data of GCaMP6m as shown. Define the intracellular fluorescence increasing between two and 2.5 standard deviations as small spikes and those increasing more than 2.5 standard deviations as large spikes.Calcium signals were observed in the mushroom body of flies. The cac knockdown flies showed more large spikes than small spikes.