in vivo calcium imaging using synaptically localized calcium sensors in drosophila melanogaster

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

For visualization of the GFP-based calcium indicators, tune the laser of a multiphoton microscope equipped with an infrared laser, and a water immersion objective installed on a vibration-isolated table, to an excitation wavelength of 920 nanometers, and install a GFP Band-pass Filter. Using the coarse Z adjustment knob, scan through the Z-axis of the brain to locate the brain region of interest.
Use the Crop function to focus the scanning on only the area of interest to minimize scan time, and rotate the scan view such that the anterior of the head is facing downwards. Then, adjust the frame size to 512 by 512 pixels, and select the region to be scanned, taking into account the calculated scan time for each frame to achieve a frame rate of at least 4 hertz.
For odor-evoked calcium transient visualization, initiate a pre-programmed macro-package capable of linking the image acquisition software in the odor delivery program, and begin the measurement in the microscope software for 6.25 seconds to establish an F0 baseline value.
In the odor delivery system, deliver a 2.5-second odor stimulus indicated here by illumination of LEDs triggered by the opening and closing of specific odor cup valves, followed by 12.5 seconds of recording at the end of the odor offset. Then, repeat the delivery for a second, and third odorant in the same manner.
To perform associative conditioning in this setup, use the computer-controlled odor delivery system to present the conditioned stimulus plus odor for 60 seconds alongside 12-90 volt electric shocks. After a 60-second break, present the conditioned stimulus minus odor alone for 60 seconds without electric shock. Measure the post-training odor-evoked calcium transience again by repeating the pre-training odor stimulation protocol, three minutes after finishing the training phase

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1. Transgenic fruit flies,&#160;Drosophila melanogaster
<ol>
	<li>Cross female virgin and male flies (raised at 25 &#176;C in 60% relative humidity on a 12 h light/dark cycle) carrying the desired Gal4 and UAS constructs, respectively, to produce flies in which specific neurons of interest express a genetically encoded calcium indicator.</li>
	<li>Age the female progeny of the above cross until they are in the range of 3-6 days post-eclosion. Female flies are preferable because of their slightly larger size.</li>
</ol>
2. Preparation of the fruit fly for in vivo calcium imaging
<ol>
	<li>Select a single female fly and anesthetize on ice for no longer than 5 min.</li>
	<li>Using fine forceps, place the fly in the imaging chamber (demonstrated in&#160;Figure 1c). Ensure the thorax and legs are in contact with the electrical wires in the bottom of the chamber and that the head lays flat. Fix the position of the fly using clear adhesive tape. 
	NOTE: This protocol requires a custom-built chamber in which the flies are fixed, with the head capsule accessible for opening, and the flies able to receive both odor stimulations to the antennae and electric shocks to the thorax and legs (Figure 1).</li>
	<li>Using a surgical scalpel blade fixed to the scalpel handle (see&#160;Table of Materials), cut a window in the tape around the head of the fly, leaving the antennae covered and only the anterior-most portion of the thorax exposed.</li>
	<li>Surround the sides and back of the head with blue-light curing glue, carefully manipulated using an insect pin held by concave-convex jaws. Set the glue using a blue light-emitting LED lamp.</li>
	<li>Check that the glue is completely set and clear any residual unhardened glue from the dorsal surface of the fly head.</li>
	<li>Apply a drop of Ringer&#39;s solution&#160;(see&#160;Table of Materials) to cover the exposed cuticle of the head.</li>
	<li>Using a very fine-bladed stab knife, cut through the cuticle. For the most efficient removal of the cuticle, first cut across the posterior of the head using the ocelli as a starting point. Then, cut up each side, medial to the eyes, to form a flap of the cuticle that can be easily torn off using forceps.</li>
	<li>Remove any excess cuticle that may block the brain region of interest.</li>
	<li>Carefully clear the dorsal surface of any trachea using fine forceps, avoiding disruption of the brain tissue itself. Remove and refresh Ringer&#39;s solution&#160;(see&#160;Table of Materials) as required to clear the area of tissue debris.</li>
	<li>Place the hypodermic odor delivery needle in a position approximately 1 cm from the head of the fly. Ensure there is nothing that could obstruct odor delivery to the antennae.</li>
	<li>At the microscope, connect the imaging chamber to the odor-delivery system via the hypodermic odor-delivery needle.</li>
	<li>To allow the fly to fully to recover from anesthesia and surgery, and to adapt to the air flow, implement a 10 min resting period at this stage.</li>
</ol>
3. In vivo calcium imaging
<ol>
	<li>Use a multiphoton microscope equipped with an infrared laser and a water immersion objective (see&#160;Table of Materials), installed on a vibration-isolated table. For the visualization of green fluorescent protein (GFP)-based calcium indicators, tune the laser to an excitation wavelength of 920 nm and install a GFP band-pass filter.</li>
	<li>Using the coarse Z adjustment knob, scan through the Z axis of the brain and locate the brain region of interest. Use the crop function to focus scanning on only this area to minimize scan time and to rotate the scan view such that the anterior of the head is facing downwards.</li>
	<li>Adjust the frame size to 512 x 512 px, scan speed to &#62; 4 Hz, and scan region (in the Y dimension) so that the neurons of interest are covered.</li>
</ol>
4. Visualization of odor-evoked calcium transients through olfactory conditioning
<ol>
	<li>Use an odor delivery system&#160;that can deliver several odor stimuli in a temporally precise manner.
	<ol>
		<li>Use an additional computer to control the device, and to communicate with the imaging microscope software to coordinate odor stimulations with image capture during experiments.</li>
		<li>Initiate a pre-programmed macro package capable of linking the image acquisition software and odor delivery program (e.g., a VBA macro package installed in the microscope control software, see&#160;Table of Materials) that is responsive to an external input trigger provided by the initiation of an odor delivery protocol in a separate program).</li>
	</ol>
	</li>
	<li>Start the measurement by monitoring &#34;pre-training&#34;/na&#239;ve odor-evoked calcium transients at a resolution of 512 x 512 px and a frame rate of 4 Hz. Deliver a 2.5 s odor stimulus flanked by additional image acquisition for 6.25 s preceding odor onset (to establish an F0&#160;baseline value) and 12.5 s after odor offset. Repeat this with a second odorant and then with a third odorant.</li>
	<li>Continue 3 min after this measurement with classical conditioning (&#34;training&#34;) the fly.
	<ol>
		<li>Select one of the odors presented in the &#34;pre-training&#34; phase to become the conditioned stimulus (CS+)&#160;odor and another to become the CS-&#160;odor. Present the CS+&#160;odor using the computer-controlled odor-deliver system for 60 s alongside twelve 90 V electric shocks.</li>
		<li>After a 60 s break, present the CS-&#160;odor alone for 60 s. Use as odorants 4-methylcyclohexanol and 3-octanol. Do not present the third odorant (e.g., 1-octen-3-ol) during this training as it is used as control only before (step 4.2.) and after (step 4.4) the training phase.</li>
	</ol>
	</li>
	<li>Measure the &#34;post-training&#34; odor-evoked calcium transients again by repeating the &#34;pre-training&#34; odor stimulation protocol (step 4.2.) 3 min after finishing the training phase (step 4.3). 
	NOTE: The timing of this step should reflect the time of interest after memory formation (e.g., carry out this step 3-4 min after the conditioning step to investigate short-term memory). Typically, flies can survive for several hours in this preparation.</li>
	<li>Save imaging files in an appropriate format (e.g., Tiff) for later image analysis.</li>
</ol>
5. Image analysis
<ol>
	<li>To analyze images, open Tiff (or similar) files in the image analysis software such as Fiji.</li>
	<li>Align the stacks using a movement correction algorithm to ensure minimal movement in the X and Y directions. Discard any recordings that show strong movement in the Z axis.</li>
	<li>Select the software tool used to mark a region of interest (ROI) around the area that is to be examined. This should be as precise as possible to limit the influence of background fluorescence.</li>
	<li>Use the respective software tool to extract temporal fluorescence intensity data as data files from the selected ROI, such that a value is generated for each frame of the recording.</li>
	<li>Open the data using a data analysis program to calculate the &#916;F/F0&#160;values for each odor trace. F0&#160;= average fluorescence in the 2 s before odor onset. &#916;F = the difference between the raw fluorescence in a given frame and F0. From these values, calculate a &#916;F/F0&#160;value for each frame, which can be plotted against time to reflect relative fluorescence throughout the odor stimulus period.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Octen-3-ol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>O5284</td>
			<td>Chemical used as odorant</td>
		</tr>
		<tr>
			<td>3-Octanol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>218405</td>
			<td>Chemical used as odorant</td>
		</tr>
		<tr>
			<td>4-Methylcyclohexanol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>153095</td>
			<td>Chemical used as odorant</td>
		</tr>
		<tr>
			<td>Bandpass filter for EGFP (525/50 nm)</td>
			<td>Carl Zeiss Microscopy GmbH, Jena, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clear adhesive tape</td>
			<td>Tesa SE, Norderstedt, Germany</td>
			<td></td>
			<td>Standard claer adhesive tape</td>
		</tr>
		<tr>
			<td>Concave-convex jaws</td>
			<td>Fine Science Tools, North Vancouver, Canada</td>
			<td>10053-09</td>
			<td>Blade Holders with concave-convex jaws</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools, North Vancouver, Canada</td>
			<td>11412-11</td>
			<td>Forceps with tip 0.1 x 0.06mm</td>
		</tr>
		<tr>
			<td>Hypodermic needle</td>
			<td>Sterican - B. Braun, Melsungenk, Germany</td>
			<td>4665120</td>
			<td>1.20x40mm</td>
		</tr>
		<tr>
			<td>Insect Minutien pins</td>
			<td>Fine Science Tools, North Vancouver, Canada</td>
			<td>26002-10</td>
			<td>Diameter 0.1mm, tip 0.0125mm</td>
		</tr>
		<tr>
			<td>Kentoflow</td>
			<td>Kent Express Dental Supplies, Gillingham, UK</td>
			<td>953683</td>
			<td>Blue light-curing glue</td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Carl Roth GmbH &#38; Co. KG, Karlsruhe, Germany</td>
			<td>656.1</td>
			<td>Standard objective slide 76 x 26 mm</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>M8410</td>
			<td>Used as diluent for odorants</td>
		</tr>
		<tr>
			<td>Mode-locked Ti-Sapphire laser Chameleon Vision 2</td>
			<td>Coherent Inc., Santa Clara, CA, USA</td>
			<td></td>
			<td>Tunable infrared femtosecond laser</td>
		</tr>
		<tr>
			<td>Multiphoton Microscope LSM 7MP equipped with BiG detectors</td>
			<td>Carl Zeiss Microscopy GmbH, Jena, Germany</td>
			<td></td>
			<td>Multiphoton microscope, multiple companies provide similar devices.</td>
		</tr>
		<tr>
			<td>Plan-Apochromat 20x (NA = 1.0) water immersion objective</td>
			<td>Carl Zeiss Microscopy GmbH, Jena, Germany</td>
			<td>421452-9900-000</td>
			<td>Objective W &#34;Plan-Apochromat&#34; 20x/1.0 DIC M27 70mm</td>
		</tr>
		<tr>
			<td>Ringer&#39;s solution</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>5mM KCl, 130mM NaCl, 2mM MgCl2, 2mM CaCl2, 5mM Hepes-NaOH, 36mM sucrose, pH = 7.4</td>
		</tr>
		<tr>
			<td>Stab knife</td>
			<td>Sharpoint, Surgical Specialties Corporation, Reading, PA, USA</td>
			<td>72-1551</td>
			<td>5.0mm Straight restricted blade depth</td>
		</tr>
		<tr>
			<td>Surgical scalpel blade</td>
			<td>Swann-Morton, Sheffield, UK</td>
			<td>303</td>
			<td>Product No. 11</td>
		</tr>
		<tr>
			<td>Surgical scalpel handle</td>
			<td>Swann-Morton, Sheffield, UK</td>
			<td>907</td>
			<td>Product No. 7S/S</td>
		</tr>
		<tr>
			<td>Visual Basics of Applicatons (VBA) software to receive a trigger from the odor-delivery device and the electric shock application device (power supply) to interact with the ZEN software from Zeiss that controls the microscope.</td>
			<td>Custom-written and available upon request</td>
			<td>n.a.</td>
			<td>n.a.</td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 1" src="/files/ftp_upload/22881/22881_Figure1.jpg" /> 
Figure 1: Construction of a mounting chamber and preparation of a fly for imaging.&#160;(a) Steps for the construction of the fly mounting chamber. The base is formed of a standard microscope slide (1) covered with a fine plastic mesh (2). Two electrical wires carrying opposing charges (3) are glued to the slide on either side. The wires are stripped and bent to cross the slide (...

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 ...

Fix a fly with its brain exposed in an imaging chamber. 
The fly expresses a genetically encoded calcium indicator, or GECI localized in the dendrites of mushroom body, or MB neurons. 
Connect the fly with electrical wires and position an odor-delivery needle near its antenna.
Add a buffer droplet on the exposed brain, position the fly under a microscope, and connect the needle to an odor-delivery system.
Excite the GECI and deliver an odor stimulus.
The odorant binds to the antenna&#39;s sensory neuron receptors, generating action potentials.
The action potentials propagate from the sensory neurons to the projection neurons, inducing neurotransmitter release and facilitating calcium influx and GECI fluorescence in the postsynaptic MB neurons.
Repeat with a second and third odorant.
Next, apply the first odor with an electric shock and the second without.
Finally, deliver the three odors without shocks. Record the fluorescence.
A reduced fluorescence in response to the shock-associated stimulus compared to the controls reveals learning-induced synaptic plasticity.

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In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

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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.

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

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 Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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

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