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 closely linked to epilepsy33,34. Previously, the behavior of Cac knockdown flies did not exhibit the severity reported in some mutants and even displayed suppressive effects in certain double mutant flies35 . The findings of this study reveal that cac knockdown flies are more prone to seizures than their wild-type counterparts. This observation corresponds with the fact that patients and other animal models carrying CACNA1A loss-of-function variants are afflicted with epilepsy28,36.&#160;Globally, the knockdown of cac could directly disrupt neuromuscular transmission in motor neurons. Although locomotion in adults and larvae was found to be non-predictive of seizure susceptibility, the contribution of motor neurons&#39; cac in seizure behavior needs to be further investigated37. Consistently, in ex vivo calcium imaging, we noticed a greater proportion of calcium events in the mushroom body of cac knockdown flies than that of the wild-type (WT) flies, which indicated a higher frequency of neuronal firing9. The mushroom body of flies is considered loosely analogous to the hippocampus and cortex in the mammalian brain. Since seizures usually occur in the hippocampus and cortex of humans, the mushroom body is used for studying seizure-related neural calcium activity in this study. In conclusion, this study established the consistency of the bang-sensitive seizure-like assay and calcium imaging in Drosophila.
In the field of calcium imaging, utilization of the calcium indicator GCaMP6 in combination with confocal microscopy enables the monitoring of alterations in calcium concentration within various contexts such as in vivo, in cell cultures, in brain slices, and in ex vivo intact brain tissue of the Drosophila model. However, it is commonly understood that the in vivo imaging of calcium fluxes within the central nervous system of Drosophila necessitates a refined surgical intervention to achieve optical access to the brain; this hindrance may inhibit the practicality and progress of this technique in individual laboratories8,16. Zebrafish is also an important animal model to study epilepsy. The in vivo calcium imaging method was also established previously36. However, only zebrafish larvae can be used for in vivo imaging recording. On the other hand, although cell culture and brain slice models can provide higher-resolution images, the integrity of the brain and its neural networks cannot be fully replicated in cell culture or brain slices due to structural disruption. The current study addresses these issues by implementing isolated intact Drosophila brain tissues for calcium imaging, thus avoiding the need for complex surgical techniques and preserving the integrity of the brain and neural networks without disruption. The ex vivo approach also yields a superior signal-to-noise ratio when compared to in vivo imaging techniques.
The present study elucidates various pivotal procedures in the protocol concerning the generation of the tub-Gal4&#62;UAS-GCaMP6m/cac-RNAi line flies. Initially, the double balancer played a crucial role in obtaining the desirable flies. Marked balancers allow researchers to follow alleles and chromosomes through complex multi-generational crosses38. Subsequently, for the efficient separation of the intact brain tissue and improved microscopic visualization, it was imperative to use Papain suspension to soften the surface fiber of the brain. Furthermore, to stabilize the brain and prevent neuronal damage during experimentation, the use of a C-sharp holder with nylon crosshairs was deemed necessary27.
In conclusion, calcium imaging applied here alongside the bang-sensitive seizure-like behavior assay in Drosophila is a powerful system for screening the epilepsy-associated genes and exploring the potential mechanism of epilepsy at the cellular level. Certainly, this technique cannot study the real-time calcium signals correlated to animal behavior like other in vivo methods.

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.

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 WT flies (88.08 &#177; 1.89 [n = 6] vs 96.50 &#177; 1.82 [n = 6]; P = 0.0093; Student&#39;s t-test, Figure 1B). As shown in Figure 2, calcium signals were observed in the mushroom body of flies. Small spikes occurred less in cac knockdown flies (tub-Gal4&#62;UAS-GCaMP6m/cac-RNAi, 2.97 &#177; 0.96 [n = 13] vs. tub-Gal4&#62;UAS-GCaMP6m, 5.33 &#177; 1.31 [n = 13]; P &#60; 0.0001; Student&#39;s t-test), whereas large spikes occurred more in cac knockdown flies (tub-Gal4&#62;UAS-GCaMP6m/cac-RNAi, 2.87 &#177; 0.63 [n = 13] vs. tub-Gal4&#62;UAS-GCaMP6m, 1.72 &#177; 0.62 [n = 13]; P &#60; 0.0001). These results indicate that the calcium signals observed in flies correlate highly with neuronal activity and showed consistency with the seizure-like behavior in cac knockdown flies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65825/65825fig01.jpg" /> 
Figure 1: Classical seizure rates and recovery time in flies. (A) Seizures occurred at a higher rate in cac knockdown flies. (B) The recovery time from seizure. <a href="https://www.jove.com/files/ftp_upload/65825/65825fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65825/65825fig02.jpg" /> 
Figure 2: Representative calcium signals in the mushroom body of flies. (A) Calcium signals in selected 5 cells in the mushroom body of flies. (B) Typical trace: Change in fluorescence (&#916;F/F) over time from an individual cell of the mushroom body in knockdown and wild-type files. (C) Global or neuronal calcium events occurring in the mushroom body of flies distinguished into small and large spikes. (D) Calcium spikes in the whole brain of knockdown flies and controls. This figure has been modified with permission from Liu et al.29. <a href="https://www.jove.com/files/ftp_upload/65825/65825fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">External solution for recording and making dissection solution.</td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>101 mM</td>
		</tr>
		<tr>
			<td>Potassium chloride&#160;</td>
			<td>3 mM</td>
		</tr>
		<tr>
			<td>Calcium chloride&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Magnesium chloride&#160;</td>
			<td>4 mM</td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic&#160;</td>
			<td>1.25 mM</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate&#160;</td>
			<td>20.7 mM</td>
		</tr>
		<tr>
			<td colspan="2">Dissection solution</td>
		</tr>
		<tr>
			<td>External solution&#160;</td>
			<td>600 &#956;L</td>
		</tr>
		<tr>
			<td>Papain suspension&#160;</td>
			<td>9 U</td>
		</tr>
		<tr>
			<td>L-lysine&#160;</td>
			<td>2 mg/mL</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of external and dissection solutions.

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

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1.3K Views.  the Second Affiliated Hospital, Guangzhou Medical University. 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.

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

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 ...

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 ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

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 ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

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. ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.