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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

Streszczenie

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Wprowadzenie

Xenopus tadpoles constitute an excellent animal model to study the normal function of the nervous system. Transparency, a fully sequenced genome1,2, and accessibility to surgical, electrophysiological and imaging techniques are unique properties of Xenopus larvae that allow investigating neuronal functions in vivo3. Some of the multiple experimental possibilities of this animal model are illustrated by the thorough studies performed on tadpole sensory and motor systems4,5,6. A particularly well-suited neuronal circuit to study many aspects of information processing at the level of synapses is the Xenopus tadpole olfactory system7. Firstly, its synaptic connectivity is well defined: olfactory receptor neurons (ORNs) project to the olfactory bulb and establish synaptic contacts with dendrites of mitral/tufted cells within glomeruli to generate odor maps. Secondly, its ORNs are continuously generated by neurogenesis throughout life to maintain the functionality of olfactory pathways8. And thirdly, because the olfactory system shows a great regenerative capability, Xenopus tadpoles are able to entirely reform their olfactory bulb after ablation9.

In this paper, we describe approaches that combine imaging of olfactory glomeruli in living tadpoles with behavioral experiments to study the functionality of olfactory pathways. The methods detailed here were used to study the functional recovery of glomerular connectivity in the olfactory bulb after olfactory nerve transection10. Data obtained in Xenopus tadpoles are representative of vertebrates since olfactory processing is evolutionary conserved.

The methods described are exemplified using X. tropicalis but they can easily be implemented in X. laevis. Despite the larger size of adult X. laevis, both species are remarkably similar during tadpole stages. The main differences reside at the genomic level. X. laevis displays poor genetic tractability, mostly determined by its allotetraploid genome and long generation time (approximately 1 year). In contrast, X. tropicalis is more amenable to genetic modifications due to its shorter generation time (5–8 months) and diploid genome. The representative experiments are illustrated for wild-type animals and three different transgenic lines: Hb9:GFP (X. tropicalis), NBT:GFP (X. tropicalis) and tubb2:GFP (X. laevis).

The methodologies outlined in the current work should be considered alongside the genetic progresses in the Xenopus field. The simplicity and easy implementation of the techniques presented makes them particularly useful for evaluating already described mutants11, as well as Xenopus lines generated by CRISPR-Cas9 technology12. We also describe a surgical procedure used to transect olfactory nerves that can be implemented in any laboratory having access to Xenopus tadpoles. The approaches used for evaluating presynaptic calcium responses and olfactory-guided behavior require specific equipment, albeit available at a moderate cost. Methodologies are presented in a simple form to promote their use in research groups and could set the bases of more complex assays either by implementing improvements or by the association to other techniques, i.e., histological or genetic approaches.

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Protokół

All procedures were approved by the animal research ethics committee at University of Barcelona.

NOTE: X. tropicalis and X. laevis tadpoles are reared according to standard methods13,14. Tadpole water is prepared by adding commercial salts (see Table of Materials) to water obtained by reverse osmosis. Conductivity is adjusted to ∼700 µS and ∼1,400 µS for X. tropicalis and X. laevis tadpoles, respectively. Larvae can be obtained either by natural mating or in vitro fertilization14. Embryos are dejellied with 2% L-cysteine prepared in 0.1x Marc's Modified Ringers (MMR). 1x MMR contains (in mM): 100 NaCl, 2 KCl, 1 MgSO4, 2 CaCl2, 5 HEPES, 0.1 EDTA, pH 7.8. Larvae are transferred after 2–3 days (stage 25) to 2 L tanks with tadpole water. When tadpoles reach stage 40 of the Nieuwkoop-Faber (NF) criteria15, they are placed in 5 L tanks and maintained at a density of 10 animals/L. Temperature is kept constant at 23–25 °C and 18–20 °C for X. tropicalis and X. laevis tadpoles, respectively. Animals found at stages 48–52 of the NF criteria are used for experiments.

1. Transection of Olfactory Nerves

  1. Prepare an anesthetizing solution of 0.02% MS-222 in 50 mL of tadpole water at room temperature.
  2. Prepare a small tank (1–2 L) with tadpole water to allow recovery of animals after surgery.
  3. Cut rectangular pieces of cellulose qualitative filter paper (4 cm x 3 cm, see Table of Materials).
  4. Wet 2 pieces of cellulose qualitative filter paper in 0.02% MS-222 solution and place them under the dissecting scope.
  5. Pick a tadpole from the tank and immerse it into the anesthetizing solution. The animal stops swimming within 2–4 min and does not react to mechanical stimuli applied at the tail level using tweezers.
  6. Place the anesthetized tadpole on the rectangular pieces of filter paper. Position the animal with its dorsal side facing upward, so brain structures can be visualized.
    1. Using vannas scissors (see Table of Materials) cut one or both olfactory nerves (depending on the type of assay to be carried out). Transect a single nerve for experiments that require an internal control of nerve injury.
    2. For behavioral experiments, transect both nerves in order to suppress all odorant information arriving to the olfactory bulb. The efficiency of sectioning of olfactory nerves can be easily observed under the dissecting scope; however, pigmentation or animal position can be limiting factors.
      NOTE: (Optional) The best way to certify the validity of the procedure is using transgenic tadpoles that express fluorescent reporters on their nervous system (see representative results). To this aim, it is necessary to use a dissection scope equipped with fluorescence (Figure 1). If only wild-type animals are available, tracing with CM-diI can be employed. Follow protocol 2 (see below) to inject a 0.5 mg/mL solution of CM-diI prepared in 0.3 M sucrose in the nasal capsule. See 16 for details on preparation and storage of CM-diI. Leakage of dye out of the principal cavity must be minimize. To this aim, it is necessary to modify the pressure of injection and the opening of micropipettes. Fluorescence at the level of the glomerular layer of the olfactory bulb becomes obvious 24 h after application of CM-diI. The present work uses labeling with CM-diI just to certify the transection procedure; however, this method can also be used to obtain morphological information of olfactory glomeruli using conventional histological procedures.
  7. Transfer animals to the recovery tank. Tadpoles should recover normal swimming within ~10 min. Perform a careful inspection for the presence of hemorrhages, which are expected in ~1% of animals subjected to surgery.
  8. Euthanize injured animals in a 0.2% MS-222 solution.

2. Labeling of Olfactory Receptor Neurons with Fluorescent Calcium Indicators

  1. Prepare a solution containing 12% Calcium Green-1-dextran (see Table of Materials), 0.1% Triton X-100, and 1 mM NaCl17. Store the solution at -20 °C or at -80 °C if it is not to be used within a month.
  2. Prepare glass pipettes with tip openings ~1–2 µm (similar diameter to microelectrodes used for patch-clamp experiments) for microinjection using a micropipette puller (see Table of Materials).
  3. Calibrate the volume of microinjections. Using distilled water, adjust pressure and injection time in order to obtain injection volumes of 0.15–0.3 µL.
    NOTE: A simple procedure consists in counting the number of pulses required to empty a pipette filled with 1 µL of water. Typical parameters are a pressure of 30 psi and 50 ms injection time.
  4. Place a pipette in the microinjector and load it with ~2 µL of calcium green-1 dextran solution.
  5. Prepare a tadpole following steps 1.1 to 1.6.
  6. Move the tip of the pipette into the principal cavity of the nasal capsule.
    NOTE: See Figure 2A describing the location of olfactory pathways in a Xenopus tadpole.
  7. Using settings obtained in 2.3, deliver a couple of puffs. Restrict dye presence to the nasal capsule.
  8. Let the tadpole rest for 2–3 min. Using a Pasteur pipette, pour drops of 0.02% MS-222 solution on the more caudal parts of the animal to avoiding drying.
  9. Transfer the animal to the recovery tank.
    NOTE: It should recover normal swimming within ~10 min. Manipulation of animals might cause injuries.
  10. Euthanize tadpoles that do not recover normal swimming behavior 15 min after injection using a 0.2% MS-222 solution.
  11. Observe fluorescence at the level of the glomerular layer of the olfactory bulb on the day after injection.

3. Preparation of Tadpoles for Live Imaging of Presynaptic Responses

  1. 24–48 h before conducting the experiment, coat 4–6 Petri dishes of 35 mm diameter with silicone elastomer (e.g., Sylgard). Once the elastomer has polymerized, fabricate a rectangular well to fit the tadpole.
    NOTE: Typical dimensions for X. tropicalis tadpoles found at NF stages 48–52 are 10 mm x 4 mm.
  2. Prepare 100 mL of a 160 µM to 1 mM amino acid solution acting as an odorant stimulus for tadpoles. The solution can contain a mixture of several amino acids: methionine, leucine, histidine, arginine and lysine. Dilute amino acids in Xenopus Ringer's solution, composed of (in mM): 100 NaCl, 2 KCl, 1 CaCl2, 2 MgCl2, 10 glucose, 10 HEPES, 240 mOsm/kg, pH 7.8. Ensure that pH is maintained at 7.8.
  3. Fill an elevated reservoir with 20 mL of the amino acid solution. Connect the reservoir with polyethylene tubing to a 28 G capillary tube (see Table of Materials) placed above the nasal capsule.
    NOTE: The capillary tube is mounted on a micromanipulator (see Table of Materials). Air bubbles must be absent from the perfusion system.
  4. Achieve temporal precision in applying the amino acid solution using the transistor-transistor logic (TTL) control of solenoid pinch valves (see Table of Materials). A stimulator is used to generate TTL pulses (see Table of Materials). Check the temporal precision to deliver the odorant solution by changing the duration of TTL pulses, i.e., 0.1 to 1 s.
  5. Fill another elevated reservoir with 100 mL of Xenopus Ringer's solution.
  6. Anesthetize a tadpole and place it under the dissecting scope (steps 1.1 to 1.6).
  7. Prepare a tadpole for imaging. If albino tadpoles are available proceed to step 3.9, otherwise remove the skin above the olfactory bulb because it contains melanocytes that impair imaging (step 3.8).
    NOTE: There are two ways to perform the experiment depending on the pigmentation of the animal. It is preferable to use albino animals. Albino strains are available for X. laevis and albino X. tropicalis lines have recently been generated by CRISPR-Cas9 12 or TALENs18.
  8. Using vannas scissors, make a lateral incision on the tadpole skin on the edge of the central nervous system. Make the cut should be made at the level of the olfactory bulb and never reaching the position of tectum, which can be easily identified by the location of the optic nerve.
  9. Pinch the cut skin using tweezers and pull it over the nervous system. Verify successful removal by the absence of melanocytes above the olfactory bulb. Keep the animal moist by pouring drops of 0.02% MS-222 solution using a Pasteur pipette.
  10. Place the tadpole into the well of the coated dish (see Table of Materials). Put a glass coverslip coated with high vacuum grease above the animal. Position the coverslip to cover the top of the tectum to the end of the tail.
  11. Ensure that the olfactory bulb and placodes remain exposed to the extracellular medium. Keep the tadpole immobile during imaging. Fill the Petri dish with Xenopus Ringer's solutioncontaining 100 µM tubocurarine (see Table of Materials) to prevent muscle contractions.
    NOTE: Tubocurarine is stored in aliquots at -80 °C no longer than 6 months.
  12. Place the dish holding the tadpole under an upright microscope. Connect the reservoir containing Xenopus Ringer's solution with the dish using polyethylene tubing (see Table of Materials) for continuous perfusion of Xenopus Ringer's solution to keep the animal alive for >1 h.
    NOTE: Mini magnetic clamps (see Table of Materials) are very useful to stably connect tubing to the dish. Perfusion and suction tubes must be located in ~180° angle.
  13. Start perfusing Xenopus Ringer's solution. Maintain the level of the solution in the dish constant throughout the experiment. Continuously evaluate tadpole viability by observing blood circulation through the vessels.

4. Live Imaging of Presynaptic Ca2+ Changes in Olfactory Glomeruli

NOTE: The imaging procedure is described for wide-field microscopy but could be easily adapted to a confocal microscope by adjusting the acquisition settings. Imaging should be carried out in an upright microscope mounted on an anti-vibration table.

  1. Visualize the tadpole with a low magnification objective, for example 5x.
  2. Move the micromanipulator axes to place the capillary delivering the odorant solution on the top of one nasal capsule forming a 90° angle with the olfactory nerve. The flow of odorant solution above the olfactory bulb should be avoided because it might cause turbulences that distort imaging.
  3. Find the olfactory bulb located ipsilaterally to the nasal capsule (subject to stimulation) using a high magnification, long working distance, water immersion objective: 60Xx, 0.9 N.A.
  4. Check the fluorescence emission by eye. Glomerular structures should be obvious (Figure 2B).
  5. Perform live acquisition with a camera suitable for calcium imaging. Define a box containing the entire olfactory bulb, typically of 256 x 256 or 512 x 512 pixels. Set the acquisition frame rate acquisition to 20–40 Hz. Adjust the gain, so that the values of basal fluorescence are ~20% of saturation. Acquire a 5 s video.
  6. Visualize the movie. Check image focus, the absence of movement artifacts and regions containing saturated pixels. Typical fluorescence values of glomerular regions should be of 5,000–20,000 a.u. if using a 16-bit camera. Proceed to the next step if imaging conditions are optimal. Repeat step 4.6 if needed to improve the image quality or adjust gain settings.
  7. Start a time-lapse acquisition to record responses evoked by olfactory stimuli.
    NOTE: Precise application of the odorant solution is controlled by TTL stimuli. A typical experiment contains a baseline period of 4 s, followed by stimulation times ranging from 0.1 to 0.5 s and a recovery period of 6–10 s.
  8. Perform repetitive stimulations of odorants for time intervals >2 min. Set the flow rate to 1–1.5 mL·min-1. Since the global perfusion is on during all experiments, locally applied amino acids are washed out.
    NOTE: The volume of solution in the dish is ~3 mL.
  9. Image analysis
    1. Detection of responses
      1. Export movies to ImageJ.
        NOTE: The goal is detecting the presence of glomerular regions responding to stimuli.
      2. Transform the raw sequence of fluorescence images to a ΔF/F0 movie. Measure relative changes in basal fluorescence according to the following relationship: (F-F0)/F0, where F0 indicates baseline fluorescence levels.
      3. Draw regions of interest (ROI) around areas showing putative fluorescence increases during stimulation and record their position in the ROI manager (Figure 2E). Draw an ROI to detect background fluorescence levels in an area devoid of glomerular structures.
    2. Quantification of responses.
      1. Place the defined ROIs in the raw sequence of fluorescence images. Obtain the mean gray value of selected ROIs for each frame. Transfer the sequence of values obtained to an analysis program (e.g., Igor Pro).
      2. Subtract background fluorescence, and then calculate ΔF/F0 changes for each ROI (Figure 2F). Plot the increases in ΔF/F0 for each one of the ROIs selected. Calculate the standard deviation of basal ΔF/F0 (before stimulation).
        NOTE: A positive response is considered if increases in ΔF/F0 obtained during stimulation are larger than 2 standard deviations of basal values.

5. Olfactory-guided Behavior Assay

NOTE: A schematic diagram of the setup for performing the assay is shown in Figure 3.

  1. Make small holes to fit 1.57 mm O.D. x 1.14 mm I.D. tubing in the upper part of each well of a 6-well dish. Insert the tubing and seal using an epoxy adhesive (see Table of Materials).
    NOTE: The modified dish can be re-used many times after thorough wash with distilled water.
  2. Prepare 50 mL of an amino acid solution containing methionine, leucine, histidine, arginine and lysine (see step 3.2 for details). The concentrations can range from 160 µM to 1 mM. Place 20 mL of the solution in an elevated reservoir.
  3. Do not feed tadpoles for at least 12 h before the assay. Take 6 tadpoles from their housing tank and place them in 2 L of clean tadpole water to minimize the exposure to odorants.
  4. Place the modified 6 wells dish on a white LED-transilluminator (Figure 3).
  5. Couple the perfusion inlets to the reservoir containing the amino acid solution using a manifold (see Table of Materials). Check the perfusion system and eliminate air bubbles. Fill the 6 wells simultaneously. Adjust the height of the reservoir to allow the delivery of ≥5 mL of odorant solution within ~30 s.
    NOTE: Wash each well 4 times with double distilled water after exposure to the odorant solution.
  6. Fill each well with 10 mL of tadpole water. Place 1 tadpole/well. Leave to rest for >3 min.
  7. Set up image acquisition. Use a conventional CCD camera that can acquire images at ≥5 Hz. Connect the camera to a computer. Here, use a Zeiss MRC5 camera controlled by Zen software but other equivalent configurations can be used. If it is necessary to increase frame rate, apply pixel binning. Images should show the whole 6-well dish.
  8. Start image acquisition such that movies contain basal (30 s), stimulus (25–35 s) and recovery (30–60 s) periods.
  9. Return animals from the 6 wells dish to tanks after imaging.
  10. Analyze movies in ImageJ using plugins such as wrMTrck19,20 that provide multiple parameters associated to motility.
  11. To prepare images for analysis, first select a well by drawing a rectangular ROI of 35 mm x 35 mm (Figure 4A). Obtain a background image by calculating the maximum projection of the whole sequence. It should display an image of the well without the tadpole.
  12. Subtract the maximum projection from the raw movie. Perform thresholding on the generated 32-bit movie and apply the wrMTrck plugin. Adjust WrMTrck parameters to reliably track animal movements. Transfer the obtained X,Y coordinates into an analysis program.
  13. Using X-Y coordinates, calculate the Euclidean distance to the odorant source (perfusion inlet in the well), by applying the following equation:
    figure-protocol-18290
    where os indicates the position of the odor source and tad indicates the tadpole position at a given time. See Figure 4A for details.

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Wyniki

In this paper, we present a combination of two complementary approaches to perform in vivo study of the functionality of the Xenopus tadpole olfactory system: i) a method for imaging presynaptic Ca2+ changes in the glomeruli of living tadpoles using a fluorescent calcium indicator, and ii) an odor guided behavioral assay that can be used to investigate the response to specific waterborne odorants. Since these approaches have been employed to evaluate the recov...

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Dyskusje

This paper describes techniques that are useful to investigate the functionality of olfactory pathways in living Xenopus tadpoles. The current protocol is particularly useful for those laboratories that work, or have access to Xenopus; however, it is also interesting for those researchers studying the cellular and molecular bases of neuronal regeneration and repair. Results obtained in Xenopus can be combined with data gathered in other vertebrate models to identify conserved mechanisms. The me...

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Ujawnienia

The authors have nothing to disclose.

Podziękowania

This work was supported by grants from El Ministerio de Economía y Competitividad (MINECO; SAF2015-63568-R) cofunded by the European Regional Development Fund (ERDF), by competitive research awards from the M. G. F. Fuortes Memorial Fellowship, the Stephen W. Kuffler Fellowship Fund, the Laura and Arthur Colwin Endowed Summer Research Fellowship Fund, the Fischbach Fellowship, and the Great Generation Fund of the Marine Biological Laboratory and the National Xenopus Resource RRID:SCR_013731 (Woods Hole, MA) where a portion of this work was conducted. We also thank CERCA Program/ Generalitat de Catalunya for institutional support. A.L. is a Serra Húnter fellow.

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Materiały

NameCompanyCatalog NumberComments
Salts for aquariums (Instant Ocean Salt)TecniplastXPSIO25R
Tricaine (Ethyl 3-aminobenzoate methanesulfonate)Sigma-AldrichE10521
Tweezers #5 (tip 0.025 x 0.005 mm)World Precision Instruments501985
Vannas Scissors (tip 0.015 x 0.015)World Precision Instruments501778
Whatman qualitative filter paperFisher ScientificWH3030917
X. laevis tubb2-GFPNational Xenopus Resource (NXR), RRID:SCR_013731NXR_0.0035
X.tropicalis NBT-GFPEuropean Xenopus Resource Center (EXRC) RRID:SCR_007164
CellTracker CM-DiIThermoFisher ScientificC-7001
Calcium Green dextran, Potassium Salt, 10,000 MW, AnionicThermoFisher ScientificC-3713
Borosilicate capillaries for microinjectionSutter InstrumentB100-75-10O.D.=1.0 mm., I.D.=0.75 mm.
PullerSutter InstrumentP-97
MicroinjectorParker InstrumentsPicospritzer III
Sylgard-184Sigma-Aldrich761028-5EA
Microfil micropipettesWorld Precision InstrumentsMF28G-5
Upright microscopeZeissAxioImager-A1
Master-8 stimulatorA.M.P.I.
CCD CameraHamamatsuImage EM
Solenoid valvesWarner InstrumentsVC-6 Six Channel system
Dow Corning High Vacuum GreaseVWR Scientific636082B
Tubocurarine hydrochlorideSigma-AldrichT2379
CCD CameraZeissMRC-5 CameraControlled by Zen software
camera lensThorlabsMVL8ML3There are multiple possibilities that should be adapted to the camera model used
Epoxy resinRS Components
ManifoldWarner InstrumentsMP-6 perfusion manifold
Micromanipulator for local delivery of solutionsNarishigeMN-153
Mini magnetic clampsWarner InstrumentsMAG-7, MAG-6
Polyethylene tubingWarner Instruments64-0755O.D.=1.57 mm., I.D.=1.14 mm.

Odniesienia

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