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 gene. Use the UAS-cac-RNAi line as the control group.</li>
	<li>After 3-5 days of eclosion, gently anesthetize the flies using the 100% CO2 (CO2 anesthesia equipment) and collect both the tub-Gal4&#62;UAS-cac-RNAi flies and the UAS-cac-RNAi flies with a brush. Transfer these flies to new, clean food vials 1 day before testing to ensure they are well-prepared.</li>
	<li>Gently anesthetize the flies using the CO2. Once anesthetized, carefully place 4-6 flies into individual fresh vials. Allow the flies to recover from anesthesia for about 1 h before proceeding with the testing. 
	NOTE: For each genotype, a minimum of five trials were tested, and each trial consisted of six vials of flies.</li>
	<li>Set up the recording equipment. Position a camera (720-1080P, 30-60 FPS at least, AF locked) on a tripod in front of a whiteboard. Adjust the camera&#39;s focus manually using an empty vial to ensure clear and accurate footage.</li>
	<li>Use a vortex mixer to perform the mechanically induced seizure-like behavior. Place each vial containing 4-6 flies onto the vortex mixer and run it at the highest setting (2800 rpm) for exactly 10 s. After the vortexing, promptly place the vial on the whiteboard.</li>
	<li>Observe the flies and record any seizure-like behavior they exhibit. 
	NOTE: Seizure-like behavior observed in flies consists of three stages: seizure (manifesting as vibrating wings), paralysis, and recovery24. The ratio of seizure flies to tested flies was defined as seizure rate.
	<ol>
		<li>Measure the recovery time as the time required after vortexing until flies regain the ability to stand upright25.</li>
	</ol>
	</li>
</ol>
2. Protocol for calcium imaging of ex vivo brain
<ol>
	<li>Establish the tub-Gal4&#62;UAS-GCaMP6m and tub-Gal4&#62;UAS-GCaMP6m/cac-RNAi line flies, respectively. Generate the flies using the Gal4/UAS binary expression system21 to allow monitoring of calcium activity in the ex vivo brain.</li>
	<li>Prepare the dissection solution as described in the provided recipe (Table 1)26.
	<ol>
		<li>Prepare the external solution as described in Table 1. Adjust to pH 7.2 with NaOH and adjust osmolarity to 250-255 mOsm/L. 
		NOTE: The external solution can be stored at 4 &#176;C for 1 week.</li>
		<li>For preparing the dissection solution, add 9 units of papain suspension and L-lysine (2 mg/mL) to 600 &#181;L of the external solution. Vortex the solution and wait 15 min until the solution is clear. 
		NOTE: It is recommended to use the dissection solution within 4 h.</li>
	</ol>
	</li>
	<li>Perfuse the external solution with oxygenated saline (95% oxygen and 5% carbon dioxide) for 5 min before use. Use the pipette to transfer about 10 &#181;L of the dissection solution to a Petri dish. This solution will provide the necessary conditions for brain dissection and imaging.</li>
	<li>Carefully dissect the brains of both the tub-Gal4&#62;UAS-GCaMP6m and tub-Gal4&#62;UAS-GCaMP6m/cac-RNAi lines using the syringe needles and microscope. Follow the established protocol for brain dissection27.</li>
	<li>Use a pipette to transfer the prepared brain into a recording dish containing 5 mL of fresh external solution and immobilize the brain using a C-sharp holder&#160;in the recording dish for 3-5 min for recovery. 
	NOTE: The C-sharp holder was made of a metal semicircle with 7 mm diameter crossed by parallel fibers 0.5 mm apart.</li>
	<li>Confocal imaging setup and acquisition
	<ol>
		<li>For confocal imaging, capture each brain with a 20x objective and xyt Scanning Mode. Identify the mushroom body neurons with an additional 4.5x digital amplification based on 20x optical amplification. 
		NOTE: Each ex vivo brain can be imaged for more than 1 h.</li>
		<li>Acquire the whole brain-GCaMP6m emission at 488 nm laser excitation with 16 &#181;w laser power. Set the scanning parameters to a speed of 400, with a pixel size of 256 pixels x 256 pixels. Use a 5.3 Hz acquisition rate and record for a duration of 3 min.</li>
	</ol>
	</li>
	<li>Identify 5-8 ROIs in every brain and analyze the fluorescence. Manually determine the cell body of mushroom body neurons as the region of interest28.
	<ol>
		<li>Label the identified neurons and measure their fluorescence intensities by ImageJ. Analyze the Fluorescence data of GCaMP6m via %&#916;F/F = (F1-F0)/(F0-B). Define the intracellular fluorescence, increasing between 2 and 2.5 standard deviations above the baseline, as small spikes, and intracellular fluorescence, increasing greater than 2.5 standard deviations above the baseline, as large spikes. Analyze the frequency of calcium spikes by Student&#39;s t-test. 
		NOTE: F0 was defined as the baseline by the mean fluorescence intensity of the initial 5 frames in ROIs, F1 as the fluorescence intensity of the given time point, and B as the background without fluorescence. Temporal physiological activities of neurons are also helpful in distinguishing them from noise signs.</li>
	</ol>
	</li>
</ol>

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

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The authors have nothing to disclose.

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><tbody><tr><td>Brushes</td><td>Panera</td><td>AAhc022-2</td><td>for handling flies</td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</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>CO&lt;sub&gt;2&lt;/sub&gt; 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 (MgCl&lt;sub&gt;2&lt;/sub&gt;)</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 (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</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 (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</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.

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

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

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

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

Research

JoVE Journal

Neuroscience

1.4K Views.  the Second Affiliated Hospital, Guangzhou Medical University. Onlangs zijn veel kandidaat-genen voor epilepsie gevonden in gentests en vervolgens gemakkelijk door dierproeven. We leveren een reeks technieken om de epilepsie-gerelateerde neurale activiteit te bestuderen, waaronder opname van hele cellen, opgewekte EPSP-opname en de nieuwe die we hier zullen introduceren, ex vivo calciumbeeldvorming. Aangezien de integriteit van de hersenen en zijn neurale netwerken niet volledig kan worden gerepliceerd in celkweek of hersenplakjes, is het belangrijkste v...

Video: Auteur in de schijnwerpers: inzichten in de technieken en bevindingen van recente ontwikkelingen in epilepsieonderzoek 

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Developmental Biology

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

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

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Biology

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Immunology and Infection

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Drosophila In Vivo Calcium Imaging: A Method for Functional Imaging of Neuronal Activity

Source: Hancock, C. E., et al. <a href="https://www.jove.com/video/60288/" target="_blank">In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes in vivo calcium imaging in Drosophila neurons using GCaMP. The featured protocol clip shows how to record changes in GCaMP fluorescence from mushroom body neurons during an olfactory conditioning experiment.

Source: Hancock, C. E., et al. In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster. J. Vis. Exp. (2019).
...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Adult

In Vivo Calcium Imaging Using Synaptically Localized Calcium Sensors in Drosophila melanogaster

Source: Hancock, C. E. et al., <a href="https://app.jove.com/v/60288">In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster.</a>&#160;J. Vis. Exp. (2019).
This video demonstrates a method for in vivo calcium imaging using synaptically localized calcium sensors in flies. It outlines the steps for preparing the flies for microscopy, conducting olfactory conditioning, and visualizing odor-induced calcium influx.

1. Transgenic fruit flies,&#160;Drosophila melanogaster

	Cross female virgin and male flies (raised at 25 &#176;C in 60% relative humidity on a 12 h ...

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

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