We thank Heidi Miller-Mommerskamp for technical help and Iveth Melissa Guatibonza Arevalo for helpful comments on the manuscript. Initial versions of the protocol were developed in the laboratory of Ralph Greenspan. This work was supported by the German research foundation (DFG), in particular through a grant FOR2705 (TP3) to IGK, and by the Simons foundation (Aimon &#8211; 414701) and the Kavli Institute for Brain and Mind (grant number #2017-954) received by SA.

All steps are performed under a stereomicroscope.
1. Preparing the holder
<ol>
	<li>Print the holder &#8216;FlyholderVJove.stl&#8217; (see Supplementary Material) with a 3D printer or have it printed using online services (Figure 1A). Both SLS (Nylon PA12) and multijet fusion (PA12) are suitable.</li>
	<li>Create the head slot.
	<ol>
		<li>Place a piece of sticky tape rectangularly on a flat surface. Cut a slice of approximately 5 mm x 1 cm. Cut the neck slot (~400 x 400 &#181;m) in the middle of the longer side of the tape using fixed parallel blades (two scalpel blades stuck together) to ensure the same width in every holder (Figure 1B).</li>
		<li>Place the tape over the flatter side of the hole in the holder on the bottom side (see Figure 1C and Figure 1D). The tape can be deformed by pushing it down with forceps ~500 &#181;m around the hole; this will further minimize hindering later fly movements.</li>
		<li>Cover the tape and the holder from the top with black nail polish to prevent the buffer from leaking out. The black nail polish will also protect the fly&#8217;s eyes from the excitation light of the microscope. Let the nail polish dry at least an hour before using the holder.</li>
		<li>Once the nail polish is dry, add ~1 &#181;L of grease in the head slot using a rolled tissue to ensure the glue will not wet the back of the fly&#8217;s head (Figure 2A). Make sure not to put grease outside of the slot that would prevent the glue from sticking.</li>
	</ol>
	</li>
	<li>Create the body slot (Figure 2B).
	<ol>
		<li>Optionally, prepare the tape that will be used to position the fly&#8217;s body (see below) in advance. Use the design shown in Figure 2B&#160;(top) to cut a piece of ~2 cm wide tape in two and cut 1.5 mm wide slices. Cut out 0.3 mm deep shoulders and body slot. Make sure that it fits the holder. 
		NOTE: The variability between fly sizes (in particular with sex, age, genotype or species) can make it necessary to adjust the tape design. If the head tends to be ill-centered, it can be helpful to add a V-shaped piece of tape temporarily over the neck slot (Figure 3). Also apply a few microliters of grease inside the neck slot and on the V-shaped tape.</li>
	</ol>
	</li>
</ol>
2. Placing the fly
NOTE: One to four days old female flies are ideal because the female head is bigger and thus easier to dissect than the male head, and younger flies have softer cuticle. For walking experiments the activity of the fly can be increased by matching the experiments with times of higher circadian activity (ZT0 or ZT11), by using saline containing glucose (such as 103 mM NaCl, 3 mM KCl, 5 mM TES, 8 mM trehalose 2 H2O, 10 mM glucose, 26 mM NaHCO3, 1 mM NaH2PO4, 2.5 mM CaCl2&#183;2 H2O, 4 mM MgCl2&#183;6 H2O), by starving the fly up to 24 h with a water only environment, and by heating the environment to ~28 &#176;C during the experiment. Clipping the wings at least one day&#160;in advance also helps to decrease attempts to fly and thus increase the frequency of walking bouts7,29,30.
<ol>
	<li>Fill a Petri dish or pipette tip box lid with ice, place a laboratory tissue on top of the ice and set the holder upside down on it.</li>
	<li>Transfer a fly on ice to paralyze it by sucking it from its vial into a tube and blowing it onto the ice (make sure the ice is not melted which would drown the fly).</li>
	<li>When the fly stops moving, use dull forceps to slide it into the holder with the neck inside the slot (the fly can be held at the basis of the wing) as in Figure 4 (left). The eyes should be at equal positions in respect to the sides of the slot (Figure 4, middle). If needed, add 1 &#181;L grease on the top of the head to prevent the glue (see next step) from reaching the back of the head and remove later.</li>
	<li>Cover the body with a tissue and some ice to make sure the fly does not move during this step and the next (Figure 4, right). Another option to prevent the legs from reaching the head is to use a piece of tape just below the head (see Figure 6).</li>
</ol>
3. Securing the head
<ol>
	<li>Place the head at a ~20&#176; angle from a fully posterior view (around the lateral axis, see Figure 5A). This is a compromise between decreasing the depth to image and on the other side maintaining the front leg free to move and minimizing the stretch of the neck.</li>
	<li>With a rolled-up tissue (Figure 5B), put UV-glue around the head while avoiding soiling the sensory area of interest (antennae, proboscis and/or eyes). For taste experiments, pull the proboscis out and add glue at its base to prevent movement. If no taste experiment is planned, the proboscis is best pushed into the head and fixed with glue to minimize movement (Figure 6).</li>
	<li>Cure the glue with UV-light for 5 s. Carefully clean the surrounding of the head with a rolled-up tissue to remove remaining liquid glue that could stick to the legs and/or soil sensory areas. 
	NOTE: The tape can be roughed with sandpaper to increase glue adhesion if necessary.</li>
	<li>Use a thin strip of tape or a rolled-up tissue to move the legs to the front (if they are not already), so they will not be damaged by the next step.</li>
</ol>
4. Positioning the body
NOTE: This step needs to be performed fast; before the fly recovers from anesthesia.
<ol>
	<li>Remove the ice container and turn the holder around. Remove the water around the fly with a tissue.</li>
	<li>Place the body slot tape (created in step 1) over the hole and gently push the fly&#8217;s body down (Figure 7). Be careful to not stretch the neck too much.</li>
</ol>
5. Sealing the hole
<ol>
	<li>Cover any remaining large holes with tape.</li>
	<li>Add ~1 &#181;L of grease to the back of the head and in the neck area to make sure no glue will wet there.</li>
	<li>With a rolled-up tissue, paint UV-glue around and on top of the tape and on the thorax (upper dorsal part of the mesonotum) to fix it. Cure the glue with UV-light for ~5 s. 
	NOTE: It is important to minimize the use of UV light as it can strongly affect the fly&#8217;s health.</li>
	<li>Carefully clean grease and uncured glue with a laboratory tissue.</li>
	<li>Put ~1 mL of saline on top of the head. Push air bubbles aside with forceps. Look for leaks by placing a coverslip over the saline and turning the holder around to check for saline on the front side. If there are any leaks, remove the saline and fix the hole (either by adding more glue or more grease). 
	NOTE: This can be a good time to pause if necessary. The fly can be offered a small piece of tissue or Styrofoam ball to walk on to prevent flailing and calm the fly down.</li>
</ol>
6. Dissecting the head
NOTE: Use sharpened forceps for the following steps. Very fine forceps are critical as dull forceps will make it more difficult to open the head cuticle and may lead to additional injuries on the fly&#8217;s head or brain. Strong magnification can help at this stage. To that aim, one can replace the oculars of the binocular microscope with 30x oculars.
<ol>
	<li>Make two cuts at the base of the central dark cuticular triangle on each side of the neck (see crosses in Figure 8A).</li>
	<li>Cut around the dark triangle and remove this part of the cuticle.</li>
	<li>The hole in the brain through which muscle 16 and the esophagus go should now be visible and move rhythmically (Video 1 presents this rhythmic movement in a fly with fluorescent muscles). Carefully pinch the top of this area to cut muscle 16 without puncturing the esophagus. If the rhythmic movement of the brain stopped, muscle 16 was likely removed, however, the movement sometimes pauses and restarts later. It is thus important to pay attention to rhythmic movements and perform this step again if necessary.</li>
	<li>Cut the remaining cuticle in small pieces and remove them carefully. Try not to pull on the cuticle too much. Instead use the forceps like a pair of scissors, to cut pieces of tissue. One can start on the medial edges, where the dark triangle was removed before and work one&#8217;s way to the sides. 
	NOTE: Pieces of cuticle can be used to gently scrape of fat bodies if present.</li>
	<li>Remove the air sacks one piece after the other by grabbing them with the forceps and pulling slowly and steadily.</li>
</ol>

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The authors have nothing to disclose.

Drosophila is one of the rare adult animals where the whole brain can be imaged during complex behaviors. Here, we present a method to prepare the fly and expose its whole brain to image ongoing whole brain activity. Several important points should be noted.
Dissecting a small animal such as D. melanogaster is challenging. The method thus requires a lot of practice and patience to master it. However, after training, the procedure takes less than 30 minutes and produces reproducible results.
The method we presented has additional limitations. First, tilting the fly head from its natural position leads to stretching the neck which could be damaging to connective tissue, nerves or muscle. Second, although the ventral subesophageal zone (SEZ) is optically accessible, it is below the semi-transparent esophagus, which decreases the intensity and resolution in this area. Finally, although the holder is out of reach in most directions, the fly still sometimes realizes its presence and pushes on it to try to escape.
Despite these limitations, the comprehensive data obtained from whole brain imaging during behavior and responses to stimuli will make it possible to decipher brain function at the level of the whole network when the animal interacts with and navigates complex, naturalistic environments.

Imaging brain activity using various techniques have deepened the understanding of brain function. In humans, brain imaging techniques have important limitations: while functional magnetic resonance imaging (fMRI) offers spatio-temporal resolution far below single neuron resolution, fast techniques such as electroencephalography (EEG) only allows indirect and partial access to the brain1. In sufficiently big animal models such as rodents, recording of fluorescent activity sensors (e.g., GCaMP) using head-mounted microscopes permits to observe brain activity while the animal is moving in its environment2. Nevertheless, these techniques currently give access only to a small portion of the brain. Head-fixed animals can be imaged more comprehensively, but coverage is still partial (e.g., the cortex surface3). It is only in small animals, such as the zebrafish larvae, C. elegans and Drosophila that the whole brain can be imaged with temporal and spatial resolution at the level of or close to single neurons4.
D. melanogaster is particularly promising because it has long been used as a genetic model organism5 and powerful genetic tools have been developed6. Complemented by the new large scale anatomical network derived from electron microscopy7, the fly could provide unique opportunities to study complex brain dynamics generated on a large scale network8. Although the cuticle is not transparent, and must thus be removed to image the brain, in vivo functional imaging has become more and more common place since the first study in 20029 and several protocols have already been published. However, these methods involve either separating the fly head from the body10, severely restricting the fly&#8217;s movements and/or responses to stimuli11,12,13,14,15, or only permitting a small part of the brain to be imaged9,16,25,26,27,17,18,19,20,21,22,23,24. To complement these nevertheless powerful approaches, we recently developed a preparation to image the whole brain during behavior and responses to various stimuli28.
Here, we build upon this study to present a method specifically developed to image the whole brain while the fly performs semi-naturalistic behavior (i.e. walking and grooming) and responds to sensory stimuli. This is achieved by using an observation holder designed to give access to the whole brain from the dorsal-posterior side, while leaving the antennae and proboscis intact, and permitting the fly to move its legs to walk (e.g., on an air-cushioned ball). Steps for dissecting the back of the head have been refined for speed, reproducibility, and to minimize their effect on the viability and mobility of the fly.

<table><tbody><tr><td>#5 forceps</td><td>FST by DUMONT</td><td>11252-30</td><td>straight tip 0.05 x 0.02 mm, Dumoxel, 11 cm long</td></tr><tr><td>#55 forceps</td><td>FST by DUMONT</td><td>11255-20</td><td>straight tip 0.05 x 0.02 mm, Inox, 11 cm long</td></tr><tr><td>30x oculars</td><td>yegren</td><td>WF30-9-30-H</td><td>WF30X/9 High Eye-point Eyepiece Wide Field View Ocular Optical Lens for Stereo Microscope or Biological Microscope 30X, 30mm without Reticle</td></tr><tr><td>AHOME/UV flashlight</td><td>Shenzhen Yijiawan Technology Co., Ltd</td><td>B07V2W9543 (ASIN)</td><td>365 nm</td></tr><tr><td>Fotoplast Gel/UV Glue</td><td>Dreve Otoplastik GmbH</td><td>44791</td><td>GHS07, GHS08</td></tr><tr><td>Gloss Finish Transparent Tape</td><td>3M Scotch</td><td></td><td></td></tr><tr><td>KIMTECH Science/Precision wipes</td><td>Kimberly-Clark Professional</td><td>7552</td><td>11 x 21 cm</td></tr><tr><td>KL 1500 LCD/Microscope light</td><td>Schott</td><td></td><td></td></tr><tr><td>Leica MS5 Microscope</td><td>Leica</td><td></td><td>WF30X/9</td></tr><tr><td>Nail Lacqueur</td><td>Opi Products Inc., N. Hollywood</td><td>6306585338</td><td>black</td></tr><tr><td>Saline: Hepes NaH2PO4 NaHCO3 MgCl2 CaCl2 NaCl KCl sucrose threalose</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Werner Dorsch GmbH</td><td>78 621; B07SXCXWFS (ASIN)</td><td>soft handle</td></tr><tr><td>Vacuum grease</td><td>Dow corning</td><td>0020080 /100 gr</td><td>Moly Kote 111 Compound Grease Grease Valve Stamp 100 g</td></tr></tbody></table>

preparing adult drosophila melanogaster for whole brain imaging during behavior and stimuli responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

The preparation described above allows observation of the whole brain under a microscope for large scale 3D imaging such as classical 2 photons or confocal microscopy, but also faster techniques such as light sheet31 and other structured illumination microscopy techniques (reviewed in32), or light field microscopy28.
The access to the whole brain while observing the behavior and maintaining functional sensory organs allows to answer several questions.
First, what is the overall brain activity when the fly is at rest, during behavior, and when it responds to stimuli? As an example, we include data obtained with a light field microscope showing brain activation during responses to stimuli and behavior. For example, in Video 2, a calcium probe was expressed in all neurons (nsyb-GAL4 and UAS-syt-GCaMP6s (left) or UAS-GCaMP6M (right)) and a puff of odor was presented. Notice how the preparation allows to get an overview of brain activity during the response to the stimulus. The powerful genetic tools in Drosophila can be used to restrict the expression of these sensors to specific neuronal subtypes. In Video 3, we restricted the expression of a calcium sensor to dopaminergic and serotoninergic neurons (TH-GAL4, DDC-GAL4 and UAS-GCaMP6M). Notice the strong synchronous activity over the brain tightly correlated with the fly walking, allowed by observing the whole brain during behavior. In addition to calcium activity, other physical or chemical signals can be imaged (using for example sensors for voltage28,33, metabolism products34,35 or specific neuromodulators36).
To understand brain activity&#8217;s role more specifically, we can ask what regions are involved in what behavior, response to stimuli or spontaneous patterns of activity. The data can indeed be used as an unbiased screen to extract functional regions using techniques such as principal component analysis and independent component analysis. Figure 9A shows different functional regions in different colors. The shape and localization of the functional regions allow to map them to anatomical templates to identify brain regions and in some cases neuron type. Furthermore, once aligned to the anatomical template, fluorescence values can be averaged in anatomical brain regions for quantitative analysis (see Figure 9B). For example, a hierarchical gaussian model applied to data in Figure 9B shows that regions are more active during walk (with a &#916;F/F median of 0.029, 95% credible interval=[0.017 0.041]) but not during groom (&#916;F/F median = -0.0049 with 95% credible interval=[-0.016 0.0059]).
Adding to the understanding depth, the simultaneous recordings of the different functional regions allowed by the preparation can be used to study the dynamical properties of the functional network. This is important because brain regions in all brains studied so far are highly recurrently interconnected and more and more studies show that even sensory areas respond to the animal&#8217;s behavioral state. Several aspects can be looked at such as functional graph properties (e.g., modules) and spatio-temporal patterns that can be fit with dynamical systems (see8 for examples).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61876/61876fig01.jpg" /> 
Figure 1: Preparing the holder (step 1).&#160;A) Holder design (view from the top). B) Neck slot preparation. top: tape design, bottom: parallel blades used to cut the neck slot. C) View of the holder from below. D) Bottom view indicating where to add the neck slot tape. <a href="https://www.jove.com/files/ftp_upload/61876/61876fig01large.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/61876/61876fig02.jpg" /> 
Figure 2: Further preparations (step 1).&#160;A) Add grease into the neck slot to prevent glue to cover the back of the head and to prevent saline leaks. Scale bars, 1 mm. B) Top: shape of the piece of tape that will be used to push the body down. Bottom: make sure the tape fits the holder. <a href="https://www.jove.com/files/ftp_upload/61876/61876fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61876/61876fig03.jpg" /> 
Figure 3: Placement of a v-shaped piece of tape to aid&#160;centering.&#160;(A) Bottom view. (B) Top view. Scale bar, 2 mm. <a href="https://www.jove.com/files/ftp_upload/61876/61876fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61876/61876fig04.jpg" /> 
Figure 4: Place the fly (step 2).&#160;Left, use two dull forceps to place the body so that the neck is in the neck slot. Middle, align the head. The eyes lie on both sides on the edges of the slot. The fly&#8217;s head is straight. Right, tuck the fly with a tissue covered with ice to keep it from moving. Scale bar, 1 mm. <a href="https://www.jove.com/files/ftp_upload/61876/61876fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61876/61876fig05.jpg" /> 
Figure 5: Fix the head (step 3).&#160;A) Ideal angle of the head. B) Add UV glue around the head, avoiding sensory areas of interest. Scale bar, 1 mm. <a href="https://www.jove.com/files/ftp_upload/61876/61876fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61876/61876fig06.jpg" /> 
Figure 6: The proboscis&#8217; tip and the antennae can be kept free from glue (step 3).&#160;The white circle indicates the area where the UV-glue has been applied. The arrow shows which parts are left free from glue for olfactory and gustatory experiments. Note the optional tape that prevents the legs from touching the glue area. Scale bar, 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61876/61876fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61876/61876fig07.jpg" /> 
Figure 7: Place the back tape (step 4.2).&#160;The tape created in step 1 (see Figure 2, left) is used to place the body and cover the large hole in the holder. Scale bar, 1 mm. <a href="https://www.jove.com/files/ftp_upload/61876/61876fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61876/61876fig08v2.jpg" /> 
Figure 8: Dissection steps (step 6).&#160;A-B) Cut on each side at the base of the dark triangle (crosses) and remove this part of the cuticle C). D) and E) remove muscle 16. Then gently remove the rest of the cuticle F), the air sacks G), and muscles. H) Dissected head. Scale bar, 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61876/61876fig08v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61876/61876fig09.jpg" /> 
Figure 9: Representative results. A) Functional regions (extracted using PCA and ICA as in28) for a fly expressing GCaMP6 pan-neuronally imaged with a light field microscope (25x, NA=0.95 with a matching f/12 micro lens array). Scale bar, 100 &#181;m. B) Average calcium activity in large brain regions during responses to stimuli and behavior (reproduced from28). <a href="https://www.jove.com/files/ftp_upload/61876/61876fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Muscle 16 movements (step 6.3). GFP was expressed in muscles. Note the pumping movement that comes from the hole just above the trachea. <a href="https://cloudflare.jove.com/files/ftp_upload/61876/Muscle16_Movie.avi" target="_blank">Please click here to download this video.</a>
Video 2: Pan-neuronal calcium activity during response to odor for two different preparations. Balsamic vinegar was puffed onto the fly. Pan-neuronal calcium activity (nsyb-GAL4 driver, UAS-syt-GCaMP6s left and UAS-GCaMP6M right) was imaged with a light field microscope (25x, NA=0.95 with a matching f/12 microlens array). Light field images were then reconstructed as described in ref.28,37. <a href="https://cloudflare.jove.com/files/ftp_upload/61876/B9_B10CombinedStacks.avi" target="_blank">Please click here to download this video.</a>
Video 3: Calcium activity in a neuron type subset during behavior. GCaMP6 was expressed in dopaminergic and serotoninergic neurons (with TH-GAL4 and DDC-GAL4). In combination with the fly genetic tools, the preparation allows to observe a strong increase in activity in many regions during walk. <a href="https://cloudflare.jove.com/files/ftp_upload/61876/Video3.avi" target="_blank">Please click here to download this video.</a>
Supplementary Material.&#160; <a href="https://cloudflare.jove.com/files/ftp_upload/61876/FlyHolderVJove.stl" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses at JoVE.com

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

preparing-adult-drosophila-melanogaster-for-whole-brain-imaging

Research

JoVE Journal

Neuroscience

6.0K Views.  Technical University of Munich. Ons protocol maakt beeldvorming van het hele vliegbrein tijdens gedrag mogelijk, wat belangrijk is om te begrijpen hoe neuronen in verschillende hersengebieden interageren om gedrag vorm te geven. In tegenstelling tot andere gepubliceerde protocollen, werd dit specifiek ontwikkeld om toegang te krijgen tot de hele hersenen terwijl de vlieg zich gedraagt en voelt door een geur, smaak of visuele stimulatie. Om een hoofdsleuf te maken.Nadat u de houder met een 3D-printer hebt afgedrukt, p...

Video: Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

Biology

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Developmental Biology

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

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 ...

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Mounting Adult Drosophila Brains: A Method to Prepare Slides for Confocal Imaging

Source: Kelly, S. M., et al. <a href="https://www.jove.com/video/56174/" target="_blank">Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains</a>. J. Vis. Exp. (2017).
This video describes a method of mounting intact brains from adult flies under a bridge slide and shows the protocol performed on immunostained brains for fluorescent microscopy.

Source: Kelly, S. M., et al. Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains. ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Adult

Ex Vivo Calcium Imaging to Study Drosophila Brain Responses to Neuropeptides

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/57701/ex-vivo-calcium-imaging-for-visualizing-brain-responses-to-endocrine-signaling-in-drosophila'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Ishimoto, H.&#160;et. al.,<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2018)</a> &#160;This video demonstrates calcium imaging to study&#160;Drosophila brain responses to neuropeptides. Neurons expressing a fluorescent calcium indicator reveal intracellular calcium dynamics and functional responses to signaling molecules.

Source:&#160;Ishimoto, H.&#160;et. al., Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila.&#160;J. Vis. Exp. ...

Neuroimaging

Live Imaging & In Vivo Tracking

Drosophila Adult Brain Dissection: A Method in Fly Neurobiology

Source: Kelly, S. M., et al. <a href="https://www.jove.com/video/56174/" target="_blank">Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains</a>. J. Vis. Exp. (2017).
This video describes how to dissect and isolate the adult Drosophila brain&#8211;a procedure necessary for visualizing the organ, which is otherwise obscured by the cuticle, eye, and tracheal tissue. The example protocol shows a detailed demonstration yielding high-quality preparations that can be used for immunostaining and imaging methods in Drosophila neurobiology.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

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