Name: Calcium Imaging of Drosophila Ex Vivo Brain for Epileptiform Analysis
Uploaded: 2023-10-13T13:00:00
Description: 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

カルシウムイメージングによる ショウジョウバエ のてんかん様イベントのモニタリング

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.

著者スポットライト:てんかん研究の最近の進歩の技術と調査結果への洞察 

Ex Vivo(エクス・ビボ )てんかんのショウ ジョウバエ モデルに対するカルシウムイメージング

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  

てんかん様解析のための ショウジョウバエ Ex vivo脳のカルシウムイメージング

まず、外用溶液を酸素化生理食塩水で5分間灌流します。ピペットで、10マイクロリットルの解剖液をペトリ皿に移します。次に、注射針と顕微鏡を使用して、麻酔をかけた確立された野生型とノックアウトハエの脳を注意深く解剖します。ピペットで、準備した脳を5ミリリットルの外用溶液を入れた記録皿に移します。Cシャープホルダーで脳を固定します。次に、各脳の共焦点画像を20倍でキャプチャします。XYTスキャンモードを使用し、さらに4.5倍のデジタル増幅でキノコ体ニューロンを識別します。レーザー励起を488ナノメートル、16マイクロワットのレーザー出力に設定して、全脳GCaMP6m発光を取得します。次に、スキャンパラメータを1400の速度、ピクセルサイズを256 x 256ピクセルに設定します。アクイジション・レートを5.3ヘルツに設定し、3分間録音します。5〜8つの関心領域の蛍光を分析します。キノコ体ニューロンの細胞体を関心領域として手動で決定します。画像 J を使用して、同定されたニューロンを標識し、その蛍光強度を測定します。GCaMP6mの蛍光データを図のように解析します。2 標準偏差から 2.5 標準偏差の間で増加する細胞内蛍光を小さなスパイクとして定義し、2.5 標準偏差を超えて増加する細胞内蛍光を大きなスパイクとして定義します。カルシウムシグナルは、ハエのキノコの体で観察されました。CACノックダウンバエは、小さなトゲよりも大きなトゲを多く示しました。

Research

JoVE Journal

Neuroscience

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 ...