Name: Seizure Activity Induced by Electroshock in Drosophila Larvae (Video) | JoVE Journal
Uploaded: 2025-06-06T16:00:00.000+00:00
Duration: 6 min 10 s
Description: Learn to identify antiseizure compounds using Drosophila larvae as a model for epilepsy research. This method evaluates therapeutic interventions by monitoring behavioral and physiological seizure responses.

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We thank the many Baines lab personnel who have jointly developed this technique over many years, and in particular, Richard Marley, who put in a good deal of effort to make this technique robust and reliable. We thank Anna Munro for drawing the larvae, which is shown in Figure 2. Work in the Baines lab that has contributed to developing this technique has been generously supported by BBSRC, MRC, and the Wellcome Trust. This work is currently supported by funding from a Wellcome Trust investigator award to R.A.B. (Grant 217099/Z/19/Z). The development of this technique also benefited from the Manchester Fly Facility, which was established through funds from the University and the Wellcome Trust (Grant 087742/Z/08/Z).

The fruit fly, Drosophila melanogaster, is used in this study (see Results section for details). This technique is best suited to wandering third-instar larvae (L3). The experimental protocol is relatively simple but requires practice to perfect. In our experience, new students require about 2 weeks to master the assay and benefit greatly from viewing other, more experienced investigators perform the assay in real-time using a camera-enabled dissection microscope or similar. The reagents and the equipment used in this study are listed in the Table of Materials.
1. Larval selection
<ol>
	<li>Collect larvae from the sides of fly vials/bottles containing standard food (5 L of water, 390 g of glucose, 360 g of maize, 250 g of yeast, 40 g of agar, 135 mL of nipagin, 15 mL of propionic acid).</li>
	<li>Only use larvae that are actively moving and have left the food to crawl up the sides of the container (wandering third instars, L3). Do not select pre-pupae, which exhibit greatly reduced locomotion.</li>
</ol>
2. Electroshock procedure
<ol>
	<li>Remove a single wandering L3 and transfer it to a small plastic Petri dish (size does not matter), and gently wash it with ddH2O to remove food residue. A small (000) paintbrush is suitable for this.</li>
	<li>Transfer the single-washed larva, using a paintbrush, to an empty plastic dish (again, size does not matter). Dry the larva with a small fragment of paper towel held with forceps. Remove excess ddH2O, but do not completely dry the larvae to avoid it sticking to the plastic dish.</li>
	<li>Allow the larva to recover for 30 s. This will facilitate easy placement of the electroshock probe (see below).</li>
	<li>View the larvae under a low-power dissection microscope (15-20x), and once normal crawling behavior resumes, gently place the electroshock probe (see step 3 for details and Figure 2A) on the anterior dorsal surface of the larva above the approximate position of the CNS (see Figure 2C). 
	NOTE: This step is critical, enough pressure must be applied to provide good conductivity between probe wire and cuticle but take care not to damage the larvae. Thus, squash the larva by about one-third to one-half of its depth.</li>
	<li>Apply a 2 s pulse of constant voltage, the strength of which has been pre-determined via a titration curve (Figure 3). Any isolated voltage stimulator is suitable here. The one used here is shown in Figure 2B.</li>
	<li>Following the electric shock, start a timer. In response to the shock, larvae initiate transitory paralysis followed by occasional spasms of body wall muscle activity and rolling behavior, halting normal crawling behavior.</li>
	<li>Stop the timer when the larva has clearly moved away from its original placement on the dish. Seizure duration, or recovery time (RT), is defined as the period between stimulus onset and resumption of normal crawling behavior (e.g., a full forward peristaltic wave that results in forward movement).</li>
	<li>At the end of each day, carefully clean the probe wires by first rinsing them in 100% ethanol followed by ddH2O. Carefully inspect the wires under magnification and, if required, use forceps to gently scrape any residue from the wires. Be very careful not to alter the distance between the two wires when doing so.</li>
</ol>
3. Electroshock probe contruction
<ol>
	<li>2&#160;x 1 m lengths of electrical wire (should be thin and flexible, i.e., rated for ~3 A) will be required. To the end of each wire, solder a 5 cm length of tungsten wire (to maximize contact, wind the tungsten wire around an exposed end of the electrical wire prior to soldering). Solder push-fit connectors (e.g., banana plugs) to the other end of the wires, suitable for easy connection to the voltage stimulator.</li>
	<li>Secure both tungsten wires to an electrode holder such that the wires are parallel to one another (Figure 2). Small sections of glass capillaries (~2 cm in length) are used to hold the wires in place under the probe lock screw.</li>
	<li>Use forceps to bend the wires such that they come to within 1-2 mm close to where they exit the wire holder. Bending tungsten wires is not easy because the wires retain a &#39;memory&#39; - thus, perseverance is required. Tips to help this process include using electrical insulating tape and/or blu-tack (or similar modeling putty) to help maintain the wires in the correct orientation/distance.</li>
</ol>
4. Probe calibration
<ol>
	<li>Ensure that there are stocks of wandering L3 of a suitable wild type (negative control) and a seizure mutant (positive control). ~100 of each will be needed.</li>
	<li>Prepare larvae, one at a time, as described above.</li>
	<li>Use the probe to apply a range of voltages to sufficient larvae of each genotype for each voltage tested. It is suggested that 0 V, 2 V, 4 V, 6 V, 8 V, 10 V, and 12 V be applied for 2 s between 10-15 larvae per voltage. Shock each larva only once.</li>
	<li>Measure the recovery time for each larva and calculate the average for each voltage step applied for both genotypes.</li>
	<li>Plot the averages on a graph and fit the data with a straight line.</li>
	<li>Select a voltage with a clear and significant difference between the control and seizure mutant. Be careful not to choose a voltage that produces an overly long recovery time; otherwise, productivity will suffer because of the long wait time for recovery. 
	NOTE: This study often used a stimulation voltage that results in a recovery time of 50-100 s for wild type and 200-300 s for parabss. Exemplar calibration curves are shown in Figure 3. It is important to use 0 V to account for the act of pressing the probe onto the dorsal surface of a larva. This will cause some degree of paralysis, which is likely a defense mechanism of the larva.</li>
</ol>
5. Execution of experiments
<ol>
	<li>Electroshock larva of the desired genotype(s) or drug exposure and measure seizure recovery time. 
	NOTE: For test larvae, an n = 20 is usually sufficient, but a power calculation based on a pilot analysis will provide a more definitive n number.</li>
	<li>Always run a negative (e.g., a wild type) and a positive (e.g., parabss) control during each experiment. The n numbers do not need to be high; n = 5 is sufficient. This will provide confidence that the assay has worked as expected (e.g., no issues with the probe or stimulator).</li>
	<li>Apply a cut-off (e.g., 300 s) to avoid overly long recovery times and only consider quantifiable seizures as those with recovery times above 30 s.</li>
</ol>
6. Drug screening
<ol>
	<li>Add drugs, dissolved in an appropriate solvent, directly to the food surface and allow to soak in (and if using ethanol, time for solvent evaporation). Or, alternatively, one can add a drug (in appropriate solvent) to melted fly food. See the Results section for details.</li>
	<li>To add the drug to the melted food, scoop out food from vials, re-melt, and as the food cools to 40 &#176;C, add the drug, mix by vortex mixer, and then repour 5 mL of melted food back into vials and allow to cool before use.</li>
	<li>Run a concentration gradient to identify the optimal drug concentration. 
	NOTE: A good starting point is to add 3 mM drug solution either&#160;directly to the food surface (200 &#181;L per standard Drosophila vial) or to make a 3 mM concentration in melted food. Adult females can be allowed to lay eggs directly in this food, or larvae can be added at selected stages as required. The solvent(s) used must be added alone to selected vials as vehicle control(s).</li>
</ol>

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<li>Parker, L., Howlett, I. C., Rusan, Z. M., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Seizure+and+epilepsy%3A+Studies+of+seizure+disorders+in+Drosophila%5BTitle%5D)+AND+%22Int+Rev+Neurobiol%22%5BJournal%5D)">Seizure and epilepsy: Studies of seizure disorders in Drosophila.</a> Int Rev Neurobiol. 99, 1-21 (2011).</li>
<li>Parker, L., Padilla, M., Du, Y., Dong, K., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+as+a+model+for+epilepsy%3A+BSS+is+a+gain-of-function+mutation+in+the+para+sodium+channel+gene+that+leads+to+seizures%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Drosophila as a model for epilepsy: BSS is a gain-of-function mutation in the para sodium channel gene that leads to seizures.</a> Genetics. 187 (2), 523-534 (2011).</li>
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<li>Baines, R. A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+development+of+central+neurons+in+the+Drosophila+embryo%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Electrophysiological development of central neurons in the Drosophila embryo.</a> J Neurosci. 18 (12), 4673-4683 (1998).</li>
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</ol>

The authors declare no competing interests.

The electroshock method to induce seizure in Drosophila larvae provides a simple, yet efficient screen to identify novel antiseizure compounds or genetic manipulations. However, because this is a qualitative assay, its main limitation is that the method cannot readily identify small effect sizes. Nevertheless, the medium throughput it allows, lending itself to screen up to ~5 compounds per week, per investigator, provides a very powerful whole animal assay. The simplicity of the technique is also suited for unde...

Epilepsy remains a significant health burden, affecting approximately 1% of the population worldwide. Even though over 30 antiseizure medications (ASMs) now exist for clinical treatment, about one-third of people with epilepsy remain drug-refractory, meaning that they do not respond well to drug treatment1,2. Available drugs are also only palliative and, as such, do not prevent epileptogenesis, nor provide a cure3. Thus, there is a critical need to identify better epilepsy treatments. A roadblock to the development of more efficacious treatments is the identification of novel drug targets. Indeed, almost all current ASMs affect similar targets: ion channels, including the voltage-gated sodium channel (Nav), and inhibitory neurotransmission mediated by &#947;-aminobutyric acid (GABA)4,5. It is generally accepted that continued use of traditional methods of drug development is unlikely to radically change this scenario.
Laboratory model animals, including, but not limited to, the fruit fly Drosophila melanogaster, and the zebrafish Danio rerio, have utility for the identification of novel ASMs6,7,8. Indeed, a PubMed search for &#39;Drosophila + seizure&#39; returns 342 results, whilst the same search for zebrafish returns 578 results (both searches were conducted on 29th Jan 2025). Whilst dwarfed by the number of similar studies in mice (~15,000), the number of studies using model systems continues to grow. These studies are possible due to the mechanistic conservation of CNS function across phyla. Moreover, induced seizures in flies and fish are effectively treated with clinically used ASMs, showing that whilst the nuances of seizure behavior may appear outwardly different, the underlying mechanisms have much in common7,9,10.
The fruit fly, Drosophila, has made many seminal contributions to understanding human biology. With respect to epilepsy, this model system provides an unparalleled genetic toolbox combined with identifiable and experimentally accessible neurons7. Moreover, the connectomes for both the larval and adult CNS have now been published, and numerous cell-specific genetic driver lines have been identified11,12. Significantly, a class of mutation was serendipitously identified whereby adult flies respond to strong mechanical stimulation by a loss of posture and seizure-like activity (e.g., wing buzzing, leg shaking, etc). This class of mutation has been termed &#39;bang-sensitive&#39;13,14,15,16. A second class of seizure mutation has since been identified that responds to increased temperature, mirroring human febrile seizures17,18. However, the experimental tractability of adult flies is somewhat reduced compared to the larval stage of this same insect model. For example, it can be difficult to drug-feed adult flies, and more invasive techniques such as electrophysiology and optogenetics can be more challenging. By contrast, the Drosophila larva eats constantly to expand its body volume by ~100-fold in just 5 days to enable pupation. Therefore, we can be confident of adequate drug-feeding in larval stages. Embryogenesis is well documented and can be accurately staged, and it has, in turn, identified key milestones in CNS development, including the first acquisition of neuronal electrical properties through to circuit formation19. Once hatched, a larva goes through 3 molts (or instars) until, on day 5, it becomes &#39;wandering&#39;, whereupon it leaves the food to find a safe place to pupate. After ~100 h of pupation, an adult fly emerges with a new body and CNS (Figure 1).
Techniques to induce seizure in adults are not well suited to larval stages. Larvae lack sensory hairs, the synchronized activation of which during mechanical stimulation can lead to a seizure. Thus, to overcome these difficulties, an electroshock technique was developed to induce seizures at the wandering larval stage. The subsequent comparative analysis of seizure induction techniques across both larvae and adults reveals that larval electroshock is far less dependent on mutation type (e.g., bang-sensitive vs. temperature). Thus, we suggest that this technique should be the preferred method for testing novel mutations where the optimal seizure induction method is unknown20. The larval electroshock technique is simple, rapid, and requires minimal equipment. This technique provides an efficient means to screen novel compounds, or genetic therapies, for antiseizure efficacy across a range of mutations that mirror the genetic diversity of human epilepsy.

<table><tbody><tr><td>Electrode holder</td><td>World Precision Instruments&amp;nbsp;</td><td>M3301</td><td></td></tr><tr><td>Glass capillaries</td><td>Harvard Instruments</td><td>GC100F-10</td><td></td></tr><tr><td>Tungsten wire (99.95%)</td><td>Goodfellow Cambridge, UK</td><td></td><td>0.1 mm diameter</td></tr><tr><td>Voltage stimulator</td><td>Digitimer Ltd, UK</td><td>DS2A mkII&amp;nbsp;</td><td>Constant Voltage Isolated Stimulator&amp;nbsp;</td></tr></tbody></table>

seizure activity induced by electroshock in drosophila larvae

Seizure Activity Induced by Electroshock in Drosophila Larvae

Epilepsy presents a significant health burden that is exacerbated by high numbers of individuals who are drug-refractory. Whilst some drug-refractory patients do respond to non-drug treatments (e.g., vagal nerve stimulation, ketogenic diet, etc.), the last resort for many is challenging and expensive surgery to provide relief from seizures. Whilst it is generally acknowledged that antiseizure medications with a broader range of targets are required, the hurdle in achieving this is the identification of novel drug targets. Genetically tractable model animals offer promise in this regard. The fruit fly, Drosophila melanogaster, has become a powerful model for investigating the mechanistic basis of, and better treatments for, seizures. Many identified fly mutations result in larvae and adults exhibiting seizure-like activity in response to strong stimulation (electrical, mechanical, and/or thermal). Many of these mutations are in genes homologous to those that contribute to human genetic epilepsies (e.g., the voltage-gated Na+ channel). It is also now possible to replace a fly gene with its human equivalent that additionally, carries a disease-related mutation. Thus, the humble fly has become an avatar to model human disease. This study describes a suitable method to use Drosophila larvae for low to medium-throughput drug screens to identify unique compounds, and their targets, that have antiseizure potential.

Numerous Drosophila mutations exhibit enhanced seizure-like behavior7,20. The genetic basis of these mutations is varied, which favorably mimics the similarly varied genetic causes of human epilepsy. Three of the most studied Drosophila mutations are parabangsenseless (parabss), julius seizure (jus), and easily-shocked (eas). The parabss mutation resu...

This protocol details the use of Drosophila larvae to identify unique antiseizure compounds for the treatment of epilepsy.

Epilepsy presents a significant health burden that is exacerbated by high numbers of individuals who are drug-refractory. Whilst some drug-refractory ...

seizure-activity-induced-by-electroshock-in-drosophila-larvae

Research

JoVE Journal

Neuroscience

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Electrophysiological Recording in the Drosophila Embryo

The video demonstrates the extracellular recording of neuronal activity from the central nervous system (CNS) of Drosophila larvae. The transected CNS is positioned in a recording chamber, and the electrical activity from a descending nerve is recorded to establish the baseline firing frequency. A test agent is then introduced, and the change in firing frequency is recorded to determine the agent's excitatory or inhibitory effect.

Extracellular Recording of Electrical Activity in the Drosophila Central Nervous System

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Forebrain Electrophysiological Recording in Larval Zebrafish

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila Adult Olfactory Shock Learning

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

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.

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Learn to identify antiseizure compounds using Drosophila larvae as a model for epilepsy research. This method evaluates therapeutic interventions by monitoring behavioral and physiological seizure responses.

Seizure Activity Induced by Electroshock in Drosophila Larvae

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