This work was supported by the Guangdong Basic and Applied Basic Research Foundation (grant no. 2022A1515111123 to Jing-Da Qiao) and plan to enhance scientific research in GMU (Jing-Da Qiao). This work was also supported by the Guangzhou Medical University Student Innovation Ability Enihancement Plan (Funding No. 02-408-2304-02038XM).

1. Protocol for bang-sensitive assay
<ol>
	<li>Establish the experimental flies by crossing the tub-Gal4 driver line with the UAS-cac-RNAi line via the Gal4/UAS system21. Collect the virgin flies of the tub-Gal4 line and the male flies of the UAS-cac-RNAi line. Then, transfer the virgin and male flies into the same vial to harvest the offspring. 
	NOTE: The tub-Gal4 driver line will allow achieving global knockdown of the cac...

Monitoring Epileptiform Events in &lt;em&gt;Drosophila&lt;/em&gt; via Calcium Imaging

<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity.">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li><li>Moisescu, D. G., Ashley, C. C., Campbell, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+aspects+of+the+calcium-sensitive+photoproteins+aequorin+and+obelin.">Comparative aspects of the calcium-sensitive photoproteins aequorin and obelin.</a> Biochim Biophys Acta. 396 (1), 133-140 (1975).</li><li>Blinks, J. R., Prendergast, F. G., Allen, D. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+calcium+signals+in+vivo:+A+powerful+tool+in+physiology+and+pharmacology.">Imaging calcium signals in vivo: A powerful tool in physiology and pharmacology.</a> Br J Pharmacol. 163 (8), 1605-1625 (2011).</li><li>Neher, E., Sakaba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+of+calcium+ions+in+the+regulation+of+neurotransmitter+release.">Multiple roles of calcium ions in the regulation of neurotransmitter release.</a> Neuron. 59 (6), 861-872 (2008).</li><li>Streit, A. K., Fan, Y. N., Masullo, L., Baines, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+of+neuronal+activity+in+drosophila+can+identify+anticonvulsive+compounds.">Calcium imaging of neuronal activity in drosophila can identify anticonvulsive compounds.</a> PLoS One. 11 (2), 0148461 (2016).</li><li>Parker, L., Howlett, I. C., Rusan, Z. M., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seizure+and+epilepsy:+Studies+of+seizure+disorders+in+drosophila.">Seizure and epilepsy: Studies of seizure disorders in drosophila.</a> Int Rev Neurobiol. 99, 1-21 (2011).</li><li>Del Valle Rodriguez, A., Didiano, D., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Power+tools+for+gene+expression+and+clonal+analysis+in+drosophila.">Power tools for gene expression and clonal analysis in drosophila.</a> Nat Methods. 9 (1), 47-55 (2011).</li><li>Liu, C. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+strategies+based+on+behavioral+and+electrophysiological+methods+for+epilepsy-related+gene+screening+in+the+drosophila+model.">Efficient strategies based on behavioral and electrophysiological methods for epilepsy-related gene screening in the drosophila model.</a> Front Mol Neurosci. 16, 1121877 (2023).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+the+odor-evoked+distributed+neural+activity+in+the+drosophila+mushroom+body.">Genetic manipulation of the odor-evoked distributed neural activity in the drosophila mushroom body.</a> Neuron. 29 (1), 267-276 (2001).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unc13b+variants+associated+with+partial+epilepsy+with+favourable+outcome.">Unc13b variants associated with partial epilepsy with favourable outcome.</a> Brain. 144 (10), 3050-3060 (2021).</li><li>Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+suppression+involving+behavioral+mutants+with+altered+nerve+excitability+in+drosophila+melanogaster.">Indirect suppression involving behavioral mutants with altered nerve excitability in drosophila melanogaster.</a> Genetics. 100 (4), 597-614 (1982).</li><li>Roemmich, A. J., Schutte, S. S., O'dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+whole-cell+recordings+in+adult+drosophila+brain.">Ex vivo whole-cell recordings in adult drosophila brain.</a> Bio Protoc. 8 (14), 2467 (2018).</li><li>Gu, H., O'dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+cell+recordings+from+brain+of+adult+drosophila.">Whole cell recordings from brain of adult drosophila.</a> J Vis Exp. (6), 248 (2007).</li><li>Qiao, J., Yang, S., Geng, H., Yung, W. H., Ke, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Input-timing-dependent+plasticity+at+incoming+synapses+of+the+mushroom+body+facilitates+olfactory+learning+in+drosophila.">Input-timing-dependent plasticity at incoming synapses of the mushroom body facilitates olfactory learning in drosophila.</a> Curr Biol. 32 (22), 4869-4880 (2022).</li><li>Liu, C. -. Q., Lin, Y. -. M., Zhang, X. -. X., Peng, R. -. C., Qiao, J. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+CACNA1A+deficiency+against+seizure+in+the+CACNA1A-CELSR2+digenic+knockdown+flies.">Protective effect of CACNA1A deficiency against seizure in the CACNA1A-CELSR2 digenic knockdown flies.</a> Research Square. , (2023).</li><li>Uchitel, O. D., Inchauspe, C. G., Urbano, F. J. D. i., Guilmi, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cav2.1+voltage+activated+calcium+channels+and+synaptic+transmission+in+familial+hemiplegic+migraine+pathogenesis.">Cav2.1 voltage activated calcium channels and synaptic transmission in familial hemiplegic migraine pathogenesis.</a> J Physiol Paris. 106 (1-2), 12-22 (2012).</li><li>Le Roux, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cacna1a-associated+epilepsy:+Electroclinical+findings+and+treatment+response+on+seizures+in+18+patients.">Cacna1a-associated epilepsy: Electroclinical findings and treatment response on seizures in 18 patients.</a> Eur J Paediatr Neurol. 33, 75-85 (2021).</li><li>Alehabib, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+molecular+spectrum+of+p/q+type+calcium+channel+cav2.1+in+epileptic+patients.">Clinical and molecular spectrum of p/q type calcium channel cav2.1 in epileptic patients.</a> Orphanet J Rare Dis. 16 (1), 461 (2021).</li><li>Li, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cacna1a+mutations+associated+with+epilepsies+and+their+molecular+sub-regional+implications.">Cacna1a mutations associated with epilepsies and their molecular sub-regional implications.</a> Front Mol Neurosci. 15, 860662 (2022).</li><li>Indelicato, E., Boesch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+genotype+to+phenotype:+Expanding+the+clinical+spectrum+of+cacna1a+variants+in+the+era+of+next+generation+sequencing.">From genotype to phenotype: Expanding the clinical spectrum of cacna1a variants in the era of next generation sequencing.</a> Front Neurol. 12, 639994 (2021).</li><li>Saras, A., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+of+the+calcium+channel+gene+cacophony+suppress+seizures+in+drosophila.">Mutations of the calcium channel gene cacophony suppress seizures in drosophila.</a> Plos Genetics. 12 (1), e1005784 (2016).</li><li>Cozzolino, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+epileptiform+activity+in+zebrafish+by+statistical-based+integration+of+electrophysiology+and+2-photon+ca2++imaging.">Evolution of epileptiform activity in zebrafish by statistical-based integration of electrophysiology and 2-photon ca2+ imaging.</a> Cells. 9 (3), 769 (2020).</li><li>Mituzaite, J., Petersen, R., Claridge-Chang, A., Baines, R. 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The calcium ion serves as a crucial second messenger, playing a pivotal role in a range of physiological and pathophysiological responses to both chemical and electrical perturbations. Furthermore, the topological element of the presynaptic P/Q channels, encoded by the human CACNA1A gene, has been identified as responsible for mediating the discharge of various neurotransmitters, including glutamate30,31,32, and is clos...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brushes</td>
			<td>Panera</td>
			<td>AAhc022-2</td>
			<td>for handling flies</td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>SP8; Zeiss, Jena, Germany.</td>
			<td>N/A</td>
			<td>for calcium imaging</td>
		</tr>
		<tr>
			<td>CO2 anesthesia machine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>for Anesthetizing the flies.</td>
		</tr>
		<tr>
			<td>C-sharp holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>handmade, for mounting the brain</td>
		</tr>
		<tr>
			<td>Culture vials</td>
			<td>Biologix</td>
			<td>51-0500</td>
			<td>2.5 cm diameter, 9.5 cm height</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>National Institutes of Health, Bethesda, MD, USA</td>
			<td>version: 2.14.0</td>
			<td>for analysis</td>
		</tr>
		<tr>
			<td>Fly morgue</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>handmade, for handling flies</td>
		</tr>
		<tr>
			<td>Fly stocks</td>
			<td>cac-RNAi</td>
			<td>27244</td>
			<td>from Bloomington Drosophila Stock Center</td>
		</tr>
		<tr>
			<td>Fly stocks</td>
			<td>GCaMP6m</td>
			<td>42750</td>
			<td>from Bloomington Drosophila Stock Center</td>
		</tr>
		<tr>
			<td>Fly stocks</td>
			<td>tub-Gal4</td>
			<td>N/A</td>
			<td>from the Sion-Frech Hoffmann Institute, Guangzhou Medical University</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>High-resolution camera</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>for recording the seizure-like behavior assay</td>
		</tr>
		<tr>
			<td>L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L5626</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride solution (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain suspension</td>
			<td>Worthington Biochemical</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>SLW1480/02D</td>
			<td>for dissection</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Thermo Scientific</td>
			<td>4640010, 4640030, 4640050, 4640060</td>
			<td>for transporting a measured volume of liquid and diseccected brain</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording dish</td>
			<td>Thermo Scientific</td>
			<td>150682- Glass Based Dish</td>
			<td>for holding the brain and calcium imaging</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>S25550</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo-binocular microscope</td>
			<td>SHANG GUANG</td>
			<td>XTZ-D</td>
			<td>for handling flies and dissection</td>
		</tr>
		<tr>
			<td>Syringe needles</td>
			<td>pythonbio</td>
			<td>HCL0693</td>
			<td>for dissection</td>
		</tr>
		<tr>
			<td>Tripod</td>
			<td>WEIFENG</td>
			<td>45634732523</td>
			<td>for recording the seizure-like behavior assay</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Lab dancer, IKA, Germany/Sigma-Aldrich</td>
			<td>Z653438</td>
			<td>for performing the seizure-like behavior assay</td>
		</tr>
		<tr>
			<td>Whiteboard</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>handmade, foam pad or paper for background</td>
		</tr>
	</tbody>
</table>

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.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

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.

Using this protocol, we found that cac knockdown flies showed significantly higher rates of seizure-like behavior than the WT flies (17.00 &#177; 2.99 [n = 6] vs 4.50 &#177; 2.03 [n = 6]; P = 0.0061; Student&#39;s t-test, Figure 1A). Most tub-Gal4&#62;UAS-cac-RNAi flies recovered within 1-5 s, while UAS-cac-RNAi flies recovered within 2 s. The recovery percentage of cac knockdown flies within 1 s was significantly lower than the ...

Watch this Scientific Journal Video about Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research at JoVE.com

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 ...

ex-vivo-calcium-imaging-for-drosophila-model-of-epilepsy