We would like to thank J. Knoblich (IMP, Vienna) and M. Gonzalez-Gaitan (University of Geneva) for Drosophila stocks, and Shinya Inou&eacute; and Christian Sardet, whose marine embryo mounting techniques were the inspiration for the development of this technique. This protocol was initially developed in Y.N. Jan's laboratory. F.R and D.Z. are supported by NIH RO1 NS059971 grant, the Pennsylvania Tobacco Settlement fund, and the Fox Chase Cancer Center.

Required Materials: Dissection stereo-microscope, Double-sided tape, standard microscope slide and coverslip, dissection forceps (size 5 or 5.5), soft-bristled brush, silicone vacuum grease, 5cc syringe, Whatman paper, confocal or epifluorescence microscope with digital camera and image acquisition software.
1. Pupal Dissection
<ol>
	<li>Set up a cross (using the appropriate combination of Gal4 line and a GFP tagged fusion protein under UAS control) or place flies from a stock you wish to image in several fresh vials at 25&deg;C.</li>
	<li>SOP cells generally begin to proliferate on the pupal thorax at eighteen hours after the onset of pupariation, we therefore select "white" pupae from the appropriate fly stock or cross. White pupae have the pupal morphology, but have an unpigmented pupal case, indicating that that have pupariated within the hour.</li>
	<li>Wait approximately 18 hours.</li>
	<li>Place a piece of double-sided tape onto the slide. Collect pupae and adhere the pupal case to the double-sided tape with the ventral side down. Grasp the edge of the operculum (the circular hatch on the anterior dorsal tip of the pupal case) with the forceps</li>
	<li>Gently lift, remove, and discard the operculum, revealing the head of the immature fly.</li>
	<li>Use the forceps to begin tearing along the side of the pupal case. Lift the midsection of the pupal case from the torn side and bring it over to the opposite side, either remove it completely or attach it to the tape, revealing the thorax and anterior portion of the abdomen. (Note: There is a gap between the developing fly and the pupal case. Be careful not to puncture the fly as the wall of the case is torn.)</li>
	<li>Begin cutting along the same side of the pupal case towards the posterior end. Once again, be careful not to puncture the fly. Pull the pupal case to the opposite side of the fly and press it onto the double-sided tape. The abdomen should now be fully exposed. Place the soft-bristled brush directly beneath the head of the fly. Once the fly is stuck to the brush and free of the pupal case, use the brush to gently lift the fly off the slide.</li>
</ol>
2. Pupal Mounting
After dissection, pupae are then mounted between slide and coverslip:
<ol start="8">
	<li>Isolated pupae removed from case can then be placed on the center of a glass slide dorsal side up.</li>
	<li>Make a square frame of Whatman paper 18X18mm leaving a 10X10 mm opening in the middle. Immerse paper in water until saturated. Place around the pupae.</li>
	<li>Using a 5 cc syringe filled with silicone vacuum grease, apply a uniform layer of grease around the Whatman frame. The grease layer should fit within the coverslip (Figure 1) and the thickness should be only a bit greater than the pupal diameter.</li>
	<li>Place a small drop of water (1 &mu;l) on the center of a 22X22 mm coverslip and place the coverslip on the above preparation such that the small water droplet contacts the surface you want to image, notum in this case). Compress gently to form a complete seal of vacuum grease and flat contact surface between the coverslip and pupal cuticle.</li>
	<li>Prep can then be imaged on inverted or upright microcopes, using either epifluorecence, confocal (laser scanning or spinning disk type) or two-photon confocal. If you're careful, adult flies can be recovered after several days.</li>
</ol>
3. Representative Results:
Using an SOP-specific Gal4 line (neuralized-Gal4) crossed to GFP tagged proteins to label the mitotic spindle (tubulin or Tau-GFP) or histone proteins (H2B-GFP), one can observe division plane of SOP cell division, length of the cell cycle, or number of mitotic divisions by using time lapse imaging 9. Other fusion proteins that target cellular organelles such as Rab-GTPases to mark different endocytic compartments (Rab5-GFP for early endosomes or Rab11 GFP for recycling endosomes) or proteins important in Notch signaling regulation (Numb-, Partner of Numb -GFP, or Sanpodo-GFP) can yield important insights into the mechanisms of asymmetric cell division and membrane protein trafficking 2,3,7,10,11.
<img src="/files/ftp_upload/2706/2706fig1.jpg" alt="Figure 1" /> 
Figure 1: Pupal dissection and mounting. A. Step-by-step images show the procedure to remove the pupal case and prepare intact pupae for mounting and live cell imaging .Pupae are removed from the vial and placed, dorsal side up, on double stick tape attached to a glass slide. First column, removal of the operculum and tearing along the side of the pupal case. Second column, removal of pupal case from thoracic and abdominal region. The free pupa is then lifted from the pupal case using a soft bristled brush. B. Mounting pupae between slide and coverslip. Pupa is placed dorsal-side up on the glass slide, surrounded by a moistened frame of Whatman paper. A continuous bead of silicone vacuum grease extruded from a 5cc syringe functions to seal the slide-coverslip combination, protecting the pupa from dessication and elevating the coverslip so it rests gently on the pupal thorax. A small drop of water (1 &mu;l) at the interface between the coverslip and thorax cuticle improves image quality significantly when using immersion objectives (water or oil). <a href="https://www.jove.com/files/ftp_upload/2706/2706fig1_large.jpg">Click here to view a larger image</a>.
<img src="/files/ftp_upload/2706/2706fig2.jpg" alt="Figure 2" /> 
Figure 2: Representative images of live SOP cells and differentiated external sensory organs taken using our pupal dissection and mounting procedure. A. Images extracted from a time series of a mitotic SOP cell expressing actin-GFP fusion protein under control of neuralized-Gal4 (neur-Gal4/UAS-actin-GFP), note cortical accumulation of actin at the cleavage furrow (1 image/every 2 minutes). B. SOP daughter cells co-expressing Partner of Numb-RFP (red) and Rab5-GFP driven by neuralized-Gal4, note the asymmetric distribution of Pon-RFP in one daughter cell (pIIb) and distribution of early endosomes in both daughter cells. C. Volume rendering of a z-series taken of differentiated external sensory organs at a late pupal stage. Cuticle structures, such as mechnosensory bristles and epidermal hairs are revealed by cuticle autofluorescence (red). In addition, we've used the MARCM system to express Lgl-GFP (driven by neuralized-Gal4) in a subset of sensory organ precursor cells (green). (These images were all acquired using a Nikon C1 scanning confocal microscope using a 60X 1.45 N.A. objective)

<ol>
	<li>Gho, M., Bellaiche, Y., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+the+Drosophila+microchaete+lineage:+a+novel+intrinsically+asymmetric+cell+division+generates+a+glial+cell.">Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell.</a> Development. 126, 3573-3584 (1999).</li><li>Roegiers, F., Younger-Shepherd, S., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+types+of+asymmetric+divisions+in+the+Drosophila+sensory+organ+precursor+cell+lineage.">Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage.</a> Nat Cell Biol. 3, 58-67 (2001).</li><li>Bellaiche, Y., Gho, M., Kaltschmidt, J. A., Brand, A. H., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frizzled+regulates+localization+of+cell-fate+determinants+and+mitotic+spindle+rotation+during+asymmetric+cell+division.">Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division.</a> Nat Cell Biol. 3, 50-57 (2001).</li><li>Emery, G., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosome+dynamics+during+development.">Endosome dynamics during development.</a> Curr Opin Cell Biol. 18, 407-415 (2006).</li><li>Roegiers, F., Younger-Shepherd, S., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bazooka+is+required+for+localization+of+determinants+and+controlling+proliferation+in+the+sensory+organ+precursor+cell+lineage+in+Drosophila.">Bazooka is required for localization of determinants and controlling proliferation in the sensory organ precursor cell lineage in Drosophila.</a> Proc Natl Acad Sci U S A. 98, 14469-14474 (2001).</li><li>Roegiers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+unanticipated+alleles+of+lethal+giant+larvae+in+Drosophila+second+chromosome+stocks.">Frequent unanticipated alleles of lethal giant larvae in Drosophila second chromosome stocks.</a> Genetics. 182, 407-410 (2009).</li><li>Tong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numb+independently+antagonizes+Sanpodo+membrane+targeting+and+Notch+signaling+in+Drosophila+sensory+organ+precursor+cells.">Numb independently antagonizes Sanpodo membrane targeting and Notch signaling in Drosophila sensory organ precursor cells.</a> Mol Biol Cell. 21, 802-810 (2010).</li><li>Jafar-Nejad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sec15,+a+Component+of+the+Exocyst,+Promotes+Notch+Signaling+during+the+Asymmetric+Division+of+Drosophila+Sensory+Organ+Precursors.">Sec15, a Component of the Exocyst, Promotes Notch Signaling during the Asymmetric Division of Drosophila Sensory Organ Precursors.</a> Dev Cell. 9, 351-363 (2005).</li><li>Karbowniczek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionarily+conserved+TSC/Rheb+pathway+activates+Notch+in+tuberous+sclerosis+complex+and+Drosophila+external+sensory+organ+development.">The evolutionarily conserved TSC/Rheb pathway activates Notch in tuberous sclerosis complex and Drosophila external sensory organ development.</a> J Clin Invest. 120, 93-102 (2010).</li><li>Coumailleau, F., Furthauer, M., Knoblich, J. A., Gonzalez-Gaitan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+Delta+and+Notch+trafficking+in+Sara+endosomes+during+asymmetric+cell+division.">Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division.</a> Nature. 458, 1051-1055 (2009).</li><li>Emery, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+rab11+endosomes+regulate+delta+recycling+and+specify+cell+fate+in+the+Drosophila+nervous+system.">Asymmetric rab11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system.</a> Cell. 122, 763-773 (2005).</li><li>Justice, N., Roegiers, F., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+giant+larvae+acts+together+with+numb+in+notch+inhibition+and+cell+fate+specification+in+the+Drosophila+adult+sensory+organ+precursor+lineage.">Lethal giant larvae acts together with numb in notch inhibition and cell fate specification in the Drosophila adult sensory organ precursor lineage.</a> Curr Biol. 13, 778-783 (2003).</li></ol>

No conflicts of interest declared.

In this video, we illustrate a technique for isolating and mounting Drosophila pupaefor live cell imaging of SOP cells on the pupal notum. Removal of pupae from the pupal case requires a steady hand, appropriate tools, and some practice, but is easy to learn. It is important that staging of pupae be done accurately, in order to ensure relative ease of dissection and catching the proliferation phase of SOP development. Once mounted, pupae will continue to develop between slide and coverslip, and in most cases, will reach the pharate adult stage, and eventually eclose. In our experience, cell can be imaged over the course of several hours. This mounting technique is not limited to observation of PNS precursor cells on the pupal thorax, but can be used to visualize any cells that are close to the cuticle surface and accessible to the microscope objective, (please note that observations are done at a stage when the pupal case can be remove without killing the pupa, usually about 10 to 14h after puparium formation). We have successfully visualized junctional proteins, cytoskeletal proteins and vesicle trafficking regulators in pupal epithelial cells (unpublished data).
The pupal mounting technique has also allowed us to develop a method to image cuticular structures in late stage pupae using the scanning confocal microscope, in order to visualize the morphology of mechanosensory bristles and epidermal hairs using the intrinsic autofluorescence of the cuticle. These images resemble scanning electron micrographs of fly cuticle, but can be acquired from live animals, and allow us to simultaneously visualize cuticular morphology and expression of GFP fluorescence 6,7,12.

<ul>
<li>Scotch double stick tape</li>
<li>Glass slide</li>
<li>18mmX18mm coverslip</li>
<li>Forceps</li>
<li>Whatman paper</li>
<li>Dow corning Silicone vacuum grease</li>
<li>5 ml syringe</li>
<li>Pipetter</li>
<li>Confocal or epifluorescence microscope</li>
</ul>

live-cell imaging of sensory organ precursor cells in intact drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Watch this Scientific Journal Video about Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae at JoVE.com

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

live-cell-imaging-sensory-organ-precursor-cells-intact-drosophila

Research

JoVE Journal

Neuroscience

12.0K Views.  Fox Chase Cancer Center. בסרטון הזה, אנו מתארים שיטת הדמיה של תא חי להתחלק סימטרית חושי אבי איברים ותאי אפידרמיס ב שלם תסיסנית גלמים

Video: Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

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.

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 ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...