<ol>
<li>Select a clean dissection dish that has not been exposed to any fixatives. </li>
<li>Dissect a wandering 3rd instar Drosophila larva in Schneider's Drosophila Medium containing Ca2+ and L-glutamine, (do not cut any nerves or damage muscle fibers Nos. 7, 6, 13 or 12). </li>
<li>Select a glass filling-pipette with a 12&nbsp;micron tip (internal diameter). </li>
<li>Using a syringe and tubing (to apply negative pressure to the pipette) ensure that the pipette tip is not obstructed. </li>
<li>Select a fine plastic filling-filament that can be inserted down the length of the glass pipette. </li>
<li>Draw ~ 1 cm of 5 mM dextran-conjugated Ca2+-indicator into the plastic filament. </li>
<li>Cut all segment nerves. </li>
<li>Support the pipette on a ramp that will allow the pipette tip to approach the ventral midline of the dissected larva. </li>
<li>Draw the cut end of a nerve to segment No.4, without pinching the nerve, into the end of the pipette (include a small amount of Schneider's medium). </li>
<li>Remove the tubing and insert the plastic filament into the pipette until the end of the filament is within 50 microns of the cut end of the nerve (avoid touching the nerve). </li>
<li>Eject sufficient Ca2+-indicator onto the nerve ending to increase the volume of the Schneider's medium by about 33% (final concentration should be &lt; 2mM). Important - This must be completed within 5 minutes of cutting the nerve. </li>
<li>Place the preparation in the dark at room temperature while the nerve loads. </li>
<li>After 40 minutes remove the Ca2+-indicator using the filament. </li>
<li>Leave the pipette in place and fill it completely with fresh Schneider's medium, as this will be used to apply stimulating pulses to the nerve. </li>
<li>Allow the Ca2+-indicator to equilibrate in the nerve for at least 60 minutes, but no more than 4 hours, before commencing Ca2+-imaging. </li>
<li>Rinse the preparation with fresh Schneider's medium every 30 minutes while it is equilibrating. </li>
<li>20 minutes before imaging replace Schneider's medium with Hemolymph-Like No.6 solution (HL6; Macleod et al. 2002; 2003). </li>
<li>L-glutamic acid or glutamate can be added to HL6 solution at 7mM to desensitize postsynaptic glutamate receptors to prevent nerve-evoked muscle contraction (Macleod et al. 2004; Reiff et al. 2002; 2005). </li>
</ol>

<ol>
	<li>Macleod, G. T., Hegstrom-Wojtowicz, M., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+calcium+signals+in+Drosophila+motor+neuron+terminals.">Fast calcium signals in Drosophila motor neuron terminals.</a> J. Neurophysiol. 88, 2659-2663 (2002).</li><li>Macleod, G. T., Suster, M. L., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+neuron+activity+in+the+Drosophila+larval+CNS+detected+with+calcium+indicators.">Single neuron activity in the Drosophila larval CNS detected with calcium indicators.</a> J. Neurosci. Methods. 127, 167-178 (2003).</li><li>Macleod, G. T., Marin, L., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicles:+test+for+a+role+in+presynaptic+calcium+regulation.">Synaptic vesicles: test for a role in presynaptic calcium regulation.</a> J. Neurosci. 24, 2496-2505 (2004).</li><li>Reiff, D. F., Thiel, P. R., Schuster, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+active+zone+density+during+long-term+strengthening+of+Drosophila+neuromuscular+junctions.">Differential regulation of active zone density during long-term strengthening of Drosophila neuromuscular junctions.</a> J. Neurosci. 22, 9399-9409 (2002).</li><li>Reiff, D. F., Ihring, A., Guerrero, G., Isacoff, E. Y., Joesch, M., Nakai, J., Borst, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+performance+of+genetically+encoded+indicators+of+neural+activity+in+flies.">In vivo performance of genetically encoded indicators of neural activity in flies.</a> J. Neurosci. 25, 4766-4778 (2005).</li></ol>

The authors have nothing to disclose.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Schneider&#x2019;s Insect Medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>must contain L-glutamine and calcium</td>
</tr>
<tr>
<td>L-glutamic acid monosodium salt hydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G1626</td>
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loading drosophila nerve terminals with calcium indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Watch this Scientific Journal Video about Loading Drosophila Nerve Terminals with Calcium Indicators at JoVE.com

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

loading-drosophila-nerve-terminals-with-calcium-indicators

Research

JoVE Journal

Biology

10.0K Views.  University of Texas Health Science Center at San Antonio (UTHSCSA). Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Video: Loading Drosophila Nerve Terminals with Calcium Indicators

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 ...

Neuroscience

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Bioengineering

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

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

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

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 ...

Neurophysiology

Imaging and Optical Techniques

Detecting Physiological Responses from Sensory Neurons in Drosophila

Source: Shankar, S., et al. <a href="https://app.jove.com/v/53392">Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging.</a> J. Vis. Exp. (2016).
This video demonstrates a method for detecting sensory neuron responses in Drosophila using ligand binding and fluorescence imaging. Ligands bind to neuron receptors, causing calcium influx. The calcium binds to indicator complexes, leading to fluorescence emission, which confirms the neuronal response to the ligand.

1. Sample Preparation

	Cross the Gal4 driver line to UAS-GCaMP5G Drosophila melanogaster flies (w;P{20XUAS-IVS-GCaMP5G}attP40). Allow the cross to grow ...

Stimulation and Modulation Techniques

Microelectrode-Mediated FM Dye Loading and Unloading in Drosophila Larvae Nerve Cells

Source: Kopke, D. L., et al., <a href="https://app.jove.com/v/57765">FM dye cycling at the synapse: comparing high potassium depolarization, electrical and channelrhodopsin stimulation.</a> J. Vis. Exp. (2018)
In this video, Drosophila larvae nerve fibers are immersed in FM dye, which binds to cell membrane lipids. Suction microelectrode and electrical pulses stimulate the endocytosis process, incorporating the FM dye into newly formed endocytic vesicles. This method, which visualizes presynaptic stages of neurotransmission, is useful for studying synaptic function and neurotransmitter release.

1. Larval Glue Dissection

	Thoroughly mix 10 parts of silicone elastomer base with 1 part of silicone elastomer curing agent from the elastomer kit.
	...