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Dual-sided Voltage-sensitive Dye Imaging of Leech Ganglia
水蛭神经节的双面电压敏感染料成像   

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eLIFE
Sep 2017

Abstract

In this protocol, we introduce an effective method for voltage-sensitive dye (VSD) loading and imaging of leech ganglia as used in Tomina and Wagenaar (2017). Dissection and dye loading procedures are the most critical steps toward successful whole-ganglion VSD imaging. The former entails the removal of the sheath that covers neurons in the segmental ganglion of the leech, which is required for successful dye loading. The latter entails gently flowing a new generation VSD, VF2.1(OMe).H, onto both sides of the ganglion simultaneously using a pair of peristaltic pumps. We expect the described techniques to translate broadly to wide-field VSD imaging in other thin and relatively transparent nervous systems.

Keywords: Voltage-sensitive dye imaging (电压敏感染料成像), Whole-brain recording (全脑记录), VoltageFluor (VoltageFluor), Microdissection (显微切割), Dye-loading process (染料加载过程)

Background

A double-sided microscope is a wide-field fluorescence imaging system consisting of a pair of microscopes precisely aligned for viewing a neuronal preparation from opposite sides and with distinct focal planes at once (Tomina and Wagenaar, 2017). By combining this optical system with a new-generation voltage-sensitive dye (VSD), VoltageFluor (Miller et al., 2012; Woodford et al., 2015), fluorescence signals that encode membrane voltages with high fidelity can be simultaneously captured from neurons at different depths. We applied this pan-neuronal recording system to the nervous system of the medicinal leech, in which we elicited fictive behaviors and quantitatively manipulated membrane potential of identifiable neurons using electrophysiological methods (Tomina and Wagenaar, 2017). Fictive behaviors were induced in an isolated nervous system: local bending was elicited by intracellular stimulation of a pressure-sensitive neuron, while swimming and crawling were elicited by extracellular stimulation of a lateral nerve of a segmented ganglion and nerves of tail brain, respectively. We were able to analyze the dynamics of almost all individual identifiable neurons within a functional unit of the leech nervous system, allowing us to construct functional maps of the roles played by these neurons in various behaviors. The imaging technique potentially is applied to other nervous systems that have multiple layers of somata such as the pedal ganglia of Aplysia.

For successful VSD imaging with a double-sided microscope, three procedures are critical: (1) dissection of the target nervous system, (2) dye loading, and (3) VSD imaging itself. These procedures were not explained in detail in our previous paper (Tomina and Wagenaar, 2017), nor in other studies using the same type of dyes (Miller et al., 2012, Moshtagh-Khorasani et al., 2013, Woodford et al., 2015, Frady et al., 2016).

As part of the dissection process, removing the sheath from the surface of the target ganglion is necessary to ensure that the dye can reach the neurons. For keeping a preparation in a healthy state, it is important to handle the preparation in an adequate way and to load the dye into cells for an appropriate length of time. During VSD imaging, the nervous system must be strictly immobilized in order to suppress motion artifact. All those steps are critical for successful whole-ganglion VSD imaging using a double-sided microscope. This protocol provides the detailed procedures to realize wide-field VSD imaging in a whole ganglion of the leech.

Materials and Reagents

  1. Dissection
    1. Microdissection container, comprising:
      1. The lid of a 35-mm plastic Petri dish (e.g., Nunclon Delta, Thermo Fisher Scientific, catalog number: 153066 )
      2. 1.5-mm thick layer of transparent PDMS (Sylgard 184 elastomer, 0.5 mg kit, Dow Corning, catalog number: 4019862 )
      3. Disk of transparent PDMS (13 mm diameter, 0.65 mm thick) with a rectangular window (1.6 x 2.6 mm) cut out of it (Sylgard 184, as above)
    2. Nylon head insect pins #6 (Emil Artl Elephant brand)
    3. Small pins (Fine Science Tools, catalog number: 26002-10 )
    4. Tungsten wires, 50 μm diameter (California Fine Wire, catalog number: MS138 )
    5. Artificial pond water (0.1 % ocean strength salt solution) (e.g., Sea salt, Instant Ocean, catalog number: SS15-10 )
    6. Nervous system of a leech
      Note: We use adult medicinal leeches Hirudo verbana (2.5-3 years old), a standard species in neuroethology field, purchased from Niagara Leeches (Niagara Falls, NY, www.leeches.bi). H. medicinalis would be equally acceptable. Leeches were maintained in an aquarium tank (250 x 500 x 300 mm) half filled with artificial pond water with aeration at 15 °C and subjected to 12:12 light:dark cycle.
    7. Coarse dissection container, comprising:
      1. Enclosure (AN-1321, Bud Industries, Digi-Key Electronics, catalog number: 377-1740-ND )
      2. Small aquarium pebbles (e.g., Imagitarium Mini White Aquarium Gravel, PetCo)
      3. Dark PDMS (Sylgard 170 silicone, 0.9 kg kit, Dow Corning, catalog number: 1696157 )

  2. Dye loading
    1. Dye-loading dish connected with an outflow capillary, comprising:
      1. The lid of a 35-mm plastic Petri dish (e.g., Nunclon Delta, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 153066 )
      2. Capillary glass, standard 1.2 x 0.68 mm, 4” (A-M Systems, catalog number: 627000 )
      3. Epoxy glue (e.g., Slow-cure 30 min Epoxy, Bob Smith Industries Inc.)
      4. Transparent PDMS (Sylgard 184 elastomer, 0.5 kg kit, Dow Corning, catalog number: 4019862 )
    2. Tube for perfusion (Masterflex C-flex tubing, Cole-Parmer Instrument, catalog number: 06424-13 )
    3. Volume-restricting well:
      A 3-mm thick slab of PDMS, 25 mm in diameter, with a 16-mm diameter hole cut out of it
    4. 1,000 μl pipette tips (e.g., VWR, catalog number: 83007-380 )
    5. 200 μl pipette tips (e.g., VWR, catalog number: 53508-783 )
    6. 10 μl pipette tips (e.g., Thermo Fisher Scientific, Thermo Scientific, catalog number: 490014-502 )
    7. 70% EtOH

  3. VoltageFluor solution
    1. VF2.1(OMe).H (provided by Evan Miller, University of California, Berkeley. Reported in Woodford et al., 2015)
      Note: An alternative product can be commercially purchased: FluoVolt Membrane Potential Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: F10488 ).
    2. Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D4540 )
    3. Pluronic acid (PowerLoadTM Concentrate 100x, Thermo Fisher Scientific, InvitrogenTM, catalog number: P10020 )
    4. Hirudo verbana physiological saline (see Recipes)
      1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
      2. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911 )
      3. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: 223506 )
      4. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M9272 )
      5. Glucose (Sigma-Aldrich, catalog number: DX0145 )
      6. HEPES (Sigma-Aldrich, catalog number: H3375 )
    5. VF2.1(OMe).H solution (see Recipes)

  4. VSD imaging
    Note: Refer to Tomina and Wagenaar (2017) for details on building a double-sided microscope. Here we describe the use of the microscope.
    1. Recording dish, comprising:
      1. Lid of a 35-mm Petri dish (e.g., Nunclon Delta, Thermo Fisher Scientific, catalog number: 153066 )
      2. PDMS substrate (Sylgard 184 elastomer, see above)
      3. Round cover glass (diameter: 18 mm, thickness: 0.2 mm, Fisher Scientific, Fisherbrand, catalog number: 12-546 )
    2. Medical dressing (Tegaderm transparent film dressing, 3M, catalog number: 9505W , e.g., Amazon)
    3. Modeling clay (Crayola, any color is fine)
    4. Lens cleaning paper (e.g., Tiffen, catalog number: EK1546027T , e.g., Adorama)
    5. Glass capillaries for intracellular recording (standard-wall 1.00 x 0.75 mm with filament, 4”, A-M Systems, catalog number: 615000 )
    6. Suction electrodes for extracellular recording, comprising:
      1. Glass capillary (standard-wall 1.20 x 0.68 mm, 4”, A-M Systems, catalog number: 627000 )
      2. Stainless steel blunt needle with Luer (Component Supply, catalog number: NE-252PL )
      3. 2.5-mm mono jack (e.g., CUI, Digi-Key Electronics, catalog number: MJ-2506 )
      4. 5-ml Luer-Lok tip Syringe (BD, catalog number: 309646 )
      5. Silver wire (200 μm diameter, California Fine Wire, catalog number: 100183 )
      6. Cast acrylic base, 2” x 0.5” x 0.25”
    7. Petroleum jelly (e.g., Vaseline)

Equipment

  1. Dissection
    1. Microsurgical knife, 5.0 mm blade (SharpPoint, Surgical specialties, catalog number: 72-1551 )
    2. Coarse dissection scissors (e.g., Heritage, model: Electricians Scissors 103C ) for cutting body wall tissue
    3. Medium dissection scissors (e.g., Natsume Seisakusho, catalog number: MB-54-1 ) for dissecting connective and nerve tissues
    4. Microdissection scissors: 2-mm cutting edge vannas spring scissors (Fine Science Tools, catalog number: 15000-03 ) for desheathing of a leech ganglion
    5. Fine forceps (e.g., Dumont #5 Dumostar, Fine Science Tools, catalog number: 11295-10 )
    6. Coarse forceps (e.g., Peer-Vigor Tweezer Swiss #5, Peer, catalog number: 57.0805 )
    7. Stereomicroscope
      Note: We use a ZEISS Stemi 2000-CS with W10x/21 eye pieces at 6.5-50x magnification for most of the dissection and a ZEISS SteREO Discovery V8 with 10x/23 eye pieces and a custom focus drive at 80x magnification for desheathing (ZEISS, models: Stemi 2000-CS and SteREO Discovery.V8 ).
    8. LED illuminator with dual gooseneck light guide (Dolan-Jenner Fiber-Lite, model: Mi-LED-US-DG )
    9. Cooling plate base for dissection:
      1. DC Power supply (30 V x 10 A DC, e.g., Yescom, model: 15SDS019-DCP3010D-09 )
      2. Peltier modules (TE Technology, model: HP-127-1.4-2.5-72P )
      3. 0.25”-thick aluminum
      4. 18-gauge stainless steel
      5. Aquarium glue

  2. Dye loading
    1. Perfusion pump head (Easy-Load II Head, Masterflex L/S, Cole-Parmer Instrument, catalog number: EW-77201-60 )
    2. Perfusion pump driver (Economy drive, Masterflex L/S, Cole-Parmer Instrument, catalog number: 07554-80 )
    3. Miniature dovetail stage (Siskiyou, catalog number: DT3-100 )
    4. Glass cutting ceramic tile (Sutter Instrument, catalog number: CTS )
    5. Pipettor 100-1,000 μl (e.g., VWR, catalog number: 89079-974 )
    6. Pipettor 20-200 μl (e.g., VWR, catalog number: 89079-970 )
    7. Pipettor 0.5-10 μl (e.g., VWR, catalog number: 89079-960 )
    8. Vortexer (e.g., VWR, model: VM-3000 , catalog number: 58816-121)
      Note: This product has been discontinued.
    9. Centrifuge (e.g., Spectrafuge, Labnet International, model: C1301-R )
    10. LED illuminator with dual gooseneck light guide (Dolan-Jenner Fiber-Lite, model: Mi-LED-US-DG )
    11. Red filter for illuminator (660-nm long-pass, 0.5” diameter, Edmund Optics, catalog number: 66-045 )
    12. Instrument for drilling a hole in dye-loading/recording dishes (e.g., a lathe such as: Jet Tools, model: BDB-1340A , catalog number: 321102AK)

  3. Optical imaging
    1. Upright fluorescent microscope (Olympus, model: BX51 )
    2. Fluorescence train of an inverted fluorescent microscope (Olympus, model: IX51 )
    3. Water-immersion 20x objective lens for top microscope (Olympus, model: XLUMPlanFLN20XW )
    4. 5x objective lens top microscope (Olympus, model: MPlan FLN )
    5. 20x objective lens for bottom microscope (Olympus, model: UCPlanFLN )
    6. Vibration isolation table (e.g., Newport, models: LW3048B-OPT or VIS3048-SG2-325A )
    7. Blue LED (LedEngin, model: LZ1-10B200 )
    8. LED controller (according to Wagenaar, 2012)
    9. CCD cameras (Photometrics, model: QuantEM 512SC )
    10. Intracellular amplifiers (A-M Systems, model: Model 1600 )
    11. Four-channel differential amplifier (A-M Systems, model: Model 1700 )
    12. Flaming/Brown micropipette puller (Sutter Instrument, model: P-97 )
    13. Data acquisition board (National Instruments, model: NI USB-6221 )

Procedure

  1. Making tools
    1. Coarse dissection container:
      1. Fill the base of the container with aquarium pebbles to about half its height.
      2. Pour black Sylgard (PDMS) into the container up to 10 mm from the top.
        Note: It is critical that the pebbles are generously covered.
      3. Leave the PDMS to set overnight at room temperature or in a lab oven set to 65 °C.
    2. Microdissection container:
      1. Pour 0.65-mm thick layer of transparent Sylgard (PDMS) into one plastic Petri dish, and a 2-mm thick layer into another.
      2. Allow the PDMS to set (as above).
      3. Use a microsurgical knife to cut a disk (13 mm in diameter) with an open window (1.6 x 2.6 mm) out of the 0.65-mm thick PDMS layer.
    3. Dye-loading dish (Figure 1):
      1. Into the center of a plastic Petri dish, drill a hole of just over 1.2 mm diameter for a capillary to fit through using a belt drive bench lathe.
      2. Heat capillary glass over a gas flame and shape it into an ‘L’ with a short leg of 7 mm. Cut the long leg to 10 mm.
      3. Insert the short leg of the L into the hole from the outside.
      4. Glue the capillary to the dish with epoxy glue.
      5. Connect the end of the capillary with a short tube connected to a short (18 mm) straight capillary. This short capillary is connected to the tubing of the perfusion pump.
      6. Pour a 2-mm-thick base layer of PDMS into the Petri dish and let it set overnight.
      7. Cut a 2-mm-diameter hole out of the PDMS directly over the capillary and verify that liquid can pass through the capillary.


        Figure 1. Double-sided dye-loading system. A. Top view of a dye-loading dish. In the center of the dish there is an outflow hole for loading dye onto the bottom surface of the ganglion. The hole is connected to capillary and tube for circulation pumping. A well formed with PDMS substrate around the hole helps reduce the required volume of VSD solution. The dish is placed on two aluminum blocks so that the dish, the capillary, and the tube are stably positioned. B. Side view of a dye-loading dish. Glass capillary inserted into the bottom hole of the dish is stabilized with epoxy glue (yellow matter). (For cleaning, the dish and tube can be separated by pulling out the non-glued part of capillary and tube.) C. Spatial arrangement of outflows and intakes of the dye-loading system. PDMS disk with a target ganglion on its center window is put in the center of the well so that the outflow from the bottom side reaches the bottom surface of the ganglion. Intake capillaries are positioned close to the edge of the well so as not to bump the PDMS disk. Top outflow is placed just above the top surface of the ganglion.

    4. Recording dish
      1. Make a hole (14 mm in diameter) in the center of the plastic dish.
        Note: This can be easily done using a laser cutter, a lathe, or a mill. We have found it difficult to achieve clean holes with a handheld drill.
      2. Place an 18 mm diameter round cover glass on the bottom of the center hole, apply PDMS around the rim of the cover glass to adhere it to the Petri dish, and leave it to set overnight.
      3. Pour transparent Sylgard (PDMS) into the dish to a thickness of 1.5 mm, again leaving it to set overnight.
      4. Cut a circle out of the PDMS slightly larger than the hole in the bottom of the dish.
      5. Completely remove any pieces of PDMS substrate from the cover glass.

  2. Dissection (Video 1: Coarse dissection, Video 2: Microdissection)

    Video 1. Isolation of the leech nervous system (a short chain of ganglia). 1. Anesthetize a leech in ice-cold saline. 2. Pin down the leech’s head and tail, using insect pins on a coarse dissection container. 3. Cut the dorsal skin along with the midline with a pair of dissection scissors. 4. Cut open the skin and pin it down. 5. Find a target ganglion for imaging. 6. Use a microsurgical knife to expose a lateral nerve root to be extracellularly recorded, if needed. 7. Dissect away the blood sinus surrounding the target ganglion. 8. Dissect away the blood sinus with the lateral nerves of the ganglion. 9. Isolate a short chain of ganglia. 10. Aspirate the isolated preparation into a pipette.

    Video 2. Double-sided desheathing of a leech ganglion. 1. Position a disk of transparent PDMS in the center of the container. 2. Transfer the ganglion into a microdissection container filled with cold saline. 3. Place the ganglion over the disk of transparent PDMS. 4. Use short pieces of tungsten wire to pin down sinus (the ganglion’s ventral side is up). 5. Stretch the connectives to apply tension to the ganglion’s sheath. 6. Zoom in on the target ganglion. 7. Remove the ventral sheath from the glial packet in the following order: the posterior, the central, and the lateral glial packets. 8. Check that the cells are not displaced or damaged. 9. Unpin the ganglion, flip the dorsal side up, then pin it down. 10. Zoom in on the ganglion. 11. Remove the dorsal sheath from the lateral glial packets (posterior to anterior). 12. Check that the cells are not displaced or damaged. 13. Unpin the ganglion, flip the ventral side up, then pin it down. 14. Check that the cells are not displaced or damaged.

    1. Place a leech in a saline-filled plastic container with an ice cube made of frozen saline and allow the leech to cool down for 10-20 min before dissection.
      Note: When anesthetized sufficiently, leeches do not show the fast reflexive twisting-like response that unanesthetized leeches display when pinned down at their head or tail.
    2. Transfer the leech to a ‘coarse dissection container’ (see ‘Materials and Reagents’) that has been kept in a fridge or freezer.
    3. Use insect pins to immobilize the leech for dissection.
      Note: Throughout the dissection, keep the container set on a cooling plate (see ‘Equipment’) and use a stereomicroscope for all procedures.
    4. Cut away tissue as needed to isolate the central nervous system of the leech (either the whole nerve cord or a short chain of ganglia).
      Note: The blood sinus surrounding the nervous system needs to be dissected away only around the ganglion targeted for imaging, not around any other ganglia.
    5. Aspirate the isolated preparation into a pipette and transfer it into a microdissection container (see ‘Materials and Reagents’) filled with saline.
    6. Place this container on a cooling plate and use a high-magnification stereomicroscope for the remaining steps.
    7. Position the disk of transparent PDMS (see ‘Materials and Reagents’) in the center of the container.
    8. Place the target ganglion over disk, ventral side up, centered over the window so that the PDMS substrate does not touch the dorsal side of the ganglion.
    9. Use short pieces of tungsten wire to pin down blood sinus tissue that surrounds the lateral nerve roots to the disk.
    10. Using micro-scissors or a microsurgical knife, carefully and cleanly remove the sheath from the ventral and dorsal surfaces of the ganglion with the following steps:
      1. Remove the ventral sheath (Figure 2A).
      2. Carefully unpin the ganglion as necessary to flip it upside down over the window in the PDMS disk, taking care not to damage any neurons in the process.
      3. Remove the dorsal sheath (Figure 2B).
      Note (very important): Throughout the procedure, keep the ganglion cool by replacing the saline in the dish with fresh cold saline every 2-3 min using plastic pipettes until ready for dye loading. Time after desheathing is critical. Desheathing should be done within 15-20 min.


      Figure 2. Desheathed ganglion. For voltage-sensitive dye loading, sheath tissue covering the target ganglion (e.g., the 10th segmental ganglion) is removed from both ventral (A) and dorsal (B) sides. ‘R’ indicates the right side of the ganglion, i.e., the animal’s right side when oriented dorsal side up. Scale bars = 100 μm.

  3. Dye loading
    1. Prepare 2 ml of VSD solution (800 nM VF2.1(OMe).H) in leech saline containing 1% pluronic acid, see Recipes) and leave it at room temperature for at least an hour in the dark.
      Note: Using freshly made cold VSD solution results in significantly compromised staining and hence a dramatic drop in signal quality. Make sure that dye loading is carried out under dim red light to avoid bleaching the dyes. 
    2. Place the ‘volume-restricting well’ into the ‘dye-loading dish’ (see ‘Materials and Reagents’), centered over the bottom outflow capillary.
    3. Position the top outflow capillary loosely over the center of the well.
    4. Fill the well with VSD solution, and dip the two intake capillaries into the solution near the edge of the well.
    5. Set both peristaltic pumps to a flow rate of about 1.1 ml/min (speed knob level 2), and verify that perfusion works well.
      Note: Make sure that no bubbles accumulate inside the outflow capillaries. If bubbles are stuck inside the capillaries, temporarily increase the pumping speed to eject the bubbles. Removing bubbles must be done before initiation of VSD staining.
    6. Transfer the PDMS disk with the ganglion still attached to it into the well in the dye-loading dish and use one or two insect pins to secure it with the ganglion hanging over the outflow capillary (Figure 1A).
      Note: The ganglion is ‘hanging’ below the disk at this time with the dorsal side facing down.
    7. Bring the top outflow capillary close to the ganglion (about 1-mm distance; Figure 1C) and use both peristaltic pumps to circulate the VSD solution (1.1 ml/min flow rate) for 8 min.
    8. Flip the PDMS disk with the ganglion attached to it upside down within the well (so the ganglion is on top of the disk and the dorsal side is facing up), taking care to keep it centered over the bottom outflow capillary.
    9. Use both peristaltic pumps to circulate the VSD solution for a further 12 min.
      Note: For well-balanced brightness on the both sides of the imaged preparation, what will be the bottom of the preparation must be stained more strongly than the top because the excitation light onto the top focus plane is brighter than that onto the bottom focus plane. The top outflow more efficiently stains a ganglion than the bottom outflow, so the choice of 8 min circulation in Step C7 and 12 min in Step C9 causes the dorsal side to be more strongly stained than the ventral side. This is by design: it compensates for the reduced excitation efficiently at what will be the bottom side of the ganglion during imaging.
    10. Gently wash the preparation with cold saline after dye loading and completely replace VSD-containing saline with normal cold saline.
    11. Clean the outflows tubes and capillaries by circulating with 70% EtOH and distilled water.
      Note: This can be done after imaging.

  4. Optical imaging
    1. Stabilize the target ganglion for imaging by tightly pinning down blood sinus tissue that surrounds the nerve roots to the PDMS disk and by sandwiching adjacent connectives between small pieces of medical dressing (Figure 3C), which must also be pinned down, to minimize any motion artifacts.
    2. Place a small amount of petroleum jelly along the rim of the cover glass of a ‘recording dish’ (see ‘Materials and Reagents’; Figure 3A).
    3. Place the disk with the ganglion in the center of the recording dish, pushing it down into the petroleum jelly.
    4. Pull blood sinus tissue around the adjacent connectives from the ganglion so that the tissue can be tightly pinned down onto the periphery PDMS substrate of the recording dish (Figure 3B).
    5. Clean the bottom surface of the cover glass with lens cleaning paper before imaging.


      Figure 3. A recording dish for double-sided VSD imaging. A. Top view of the center area of recording dish (top) and a schematic frontal section view (bottom). Before placing a PDMS disk with a preparation into the dish, petroleum jelly is applied to the periphery of the cover glass. B. Top view of PDMS disk with a preparation set on the cover glass (top) and a schematic frontal section view (bottom). To minimize motion artifacts, tension is created by pinning the blood sinus tissues around the adjacent ganglia down onto the PDMS substrate of the outer area of the dish. C. An adjacent connective sandwiched between two pieces of medical dressing (top) and a schematic frontal section view (bottom). The pieces of medical dressing are pinned down onto the PDMS disk.

    6. Place the recording dish on the stage of a double-sided microscope (Figures 4A and 4B).
    7. Adjust the orientation of preparation using top (upright) microscope under red light exposure.
    8. Prepare for extracellular and/or intracellular recording by positioning electrodes near target nerves or cell bodies (Figure 4C).
      Note: Time interval between the end of dye-loading and the beginning of VSD imaging is typically 20-25 min. Spending much more time here may negatively impact the health of the ganglion.


      Figure 4. Double-sided microscope. A. Overview of a double-sided microscope. The fluorescence train of an Olympus BX upright microscope is mounted with a custom focus rack on top of the body of an Olympus IX inverted microscope. Images are acquired with two CCD cameras. B. Top and bottom 20x objectives precisely aligned for dual-sided VSD imaging. A recording dish is put in the cutout (white) of the plastic stage. Sticky clay is used for fixing the position of the dish. C. Simultaneous electrophysiology and double-sided VSD imaging. The top side of the preparation is accessible to both intracellular and extracellular electrodes.

    9. Aspirate target nerves into suction electrodes and/or impale target cells.
    10. Bring top and bottom surfaces of the ganglion coarsely into focus using top and bottom objectives.
    11. Adjust the brightness of images to obtain the best brightness but not to saturate the CCD cameras using the intensity control of the LED controller or the aperture stop of the microscope.
    12. Determine the final focus for top and bottom imaging.
      Note (important): Throughout all these preparations, frequently replace saline with cold saline.
    13. Start VSD imaging/electrophysiological trial using VScope (Wagenaar, 2017).
      Note: Make sure that optical imaging is carried out in dark surroundings.
    14. After finishing the VSD imaging experiment, acquire snapshots of both top and bottom aspects of the ganglion at different focusing depths for later creation of ‘focus-stacked’ images of the ganglion (Figure 5). These will be used for drawing for Regions of Interest (ROIs) in VScope.


      Figure 5. Visualizing the ganglion. A. Single snapshot image of the ventral aspect of the 10th ganglion as captured by top camera. B. The same image after processing with adaptive contrast enhancement (ACE) filter. C. Focus-stacked image constructed from multiple snapshots of the ventral aspect at different depths. D. The same image after processing with ACE filter. Scale bars = 100 μm.

Data analysis

  1. Even during an ongoing experiment, VScope (Wagenaar, 2017) can be used to check if the acquired optical signals show neuronal activity (spontaneous activity, stimulus-induced response, or fictive behavior). The rest of the analysis is usually performed after the end of an experiment.
  2. Draw ROIs in focus-stacked images using VScope’s ‘adaptive contrast enhancement’ filter (Figure 5), and then transfer ROI information to individual trials.
  3. Load the data into Octave using the functions provided with VScope.
  4. Clean up VSD imaging data using motion correction and debleaching algorithms (Tomina and Wagenaar, 2017).
  5. Perform further statistical analysis as relevant to your experiment, e.g., using Octave (Figure 6).
  6. Check that intracellular recording matches VSD signals from selected neurons to confirm the reliability of optical recording. Also check whether stereotypical motor patterns are observed in representative neurons like ventral/dorsal motor neurons (DI1, VI2, DE3, and VE4) especially when experiments involve fictive behaviors (Figure 6).


    Figure 6. Double-sided VSD imaging. A and B. Dual surface images (ventral in A; dorsal in B) simultaneously captured with two CCD cameras. Scale bars = 100 μm. C. Selected electrophysiological and VSD traces during fictive swimming: Extracellular recording from a nerve root in a posterior segment (DP nerve of 13th ganglion) showing rhythmic dorsal excitatory motor neuron bursts; Intracellular recording and simultaneous optical signal from left AE cell (a motor neuron, marked with *) show matching membrane potential oscillations; VSD signals from the ventral surface: bilateral AE cells and Retzius cells (a neuromodulatory neuron); VSD signals from the dorsal surface: dorsal and ventral inhibitory and excitatory motor neurons DI-1, VI-2, DE-3, and VE-4. Scale bars = 1 sec for time, 10 mV for membrane potential and 0.2% ΔF/F for fluorescence signals.

Note: Sample data, codes and detailed application manual are available in Dryad Digital Repository (https://doi.org/10.5061/dryad.m20kh).

Notes

  1. Dissection should be done quickly and efficiently (typically 10-30 min for the isolation of the leech nervous system, 15-20 min for double-sided desheathing), and saline should be replaced with cold fresh saline frequently (every 2-3 min) with a pipette, because the health of the leech nervous system is critical for the success of experiments.
  2. To suppress motion artifact from contractile tissue within sheath around the nerve cords, connectives and lateral nerves should be physically stretched by pinning down the remaining blood sinus onto PDMS substrate after dye loading. If non-negligible motion artifact is observed during imaging, the strength of its stretch should be increased.
  3. Stocks of VF2.1 (OMe).H dissolved in DMSO should be kept in the refrigerator at 4 °C. To maintain the quality of staining condition, do not keep the dye at room temperature or in the deep freezer.
    Note: According to a manual of a commercial product of VoltageFluor (FluoVolt Membrane Potential Kit), this product should be stored at 2-8 °C.
  4. We qualitatively assess how well our specimens were stained using the fluorescent images and signals obtained during the experiment (examples shown in Figures 5 and 6). This is presently the only way to confirm that the staining process went well. Looking for visual changes in a ganglion during dye loading does not provide reliable information regarding the quality of staining.
    Note: We recommend that beginners of VSD imaging in the leech first practice to obtain consistent results between intracellular recordings and VSD signals by targeting a Retizus cell or any other cells that are easy to impale.

Recipes

  1. Hirudo verbana physiological saline

    Note: Adjust pH to 7.4 with NaOH or HCl.
  2. 800 nM VF2.1(OMe).H solution (1 ml)
    1. Aliquot 8 μl of 100 μM VF2.1(OMe).H (stock solution in DMSO, kept in the refrigerator) in a micro-tube and keep them in the refrigerator in advance
    2. Mix this with 10 μl of pluronic acid
    3. Add leech saline to make its final volume 1 ml and vortex it
    Note: This recipe applies to commercially available VoltageFluor dye.

Acknowledgments

We thank Evan Miller for supplying the VF2.1(OMe).H dye; Annette Stowasser for her role in developing a prototype of the double-sided microscope and many helpful conversations; Angela Bruno for useful discussions regarding data analysis; Ng Cai Tong for reading and checking the manuscript. This work was supported by the Burroughs Welcome Fund through a Career Award at the Scientific Interface and by the National Institute of Neurological Disorders and Stroke through grant R01 NS094403 (both to DAW). YT was supported by JSPS Overseas Research Fellowships. This protocol was adapted from procedures published in Tomina and Wagenaar (2017). The authors of this work declare no conflicts of interest.

References

  1. Frady, E. P., Kapoor, A., Horvitz, E. and Kristan, W. B., Jr. (2016). Scalable semisupervised functional neurocartography reveals canonical neurons in behavioral networks. Neural Comput 28(8): 1453-1497.
  2. Miller, E. W., Lin, J. Y., Frady, E. P., Steinbach, P. A., Kristan, W. B., Jr. and Tsien, R. Y. (2012). Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires. Proc Natl Acad Sci U S A 109(6): 2114-2119.
  3. Moshtagh-Khorasani, M., Miller, E. W. and Torre, V. (2013). The spontaneous electrical activity of neurons in leech ganglia. Physiol Rep 1(5): e00089.
  4. Tomina, Y. and Wagenaar, D. A. (2017). A double-sided microscope to realize whole-ganglion imaging of membrane potential in the medicinal leech. Elife 6.
  5. Wagenaar, D. A. (2012). An optically stabilized fast-switching light emitting diode as a light source for functional neuroimaging. PLoS One 7(1): e29822.
  6. Wagenaar, D. A. (2017). VScope – data acquisition and analysis for voltage-sensitive dye imaging using multiple cameras and electrophysiology. Journal of Open Research Software 5: 23.
  7. Woodford, C. R., Frady, E. P., Smith, R. S., Morey, B., Canzi, G., Palida, S. F., Araneda, R. C., Kristan, W. B., Jr., Kubiak, C. P., Miller, E. W. and Tsien, R. Y. (2015). Improved PeT molecules for optically sensing voltage in neurons. J Am Chem Soc 137(5): 1817-1824.

简介

在这个协议中,我们介绍了一种有效的方法,用于Tomina和Wagenaar(2017)中使用的电压敏感染料(VSD)加载和水蛭神经节成像。 解剖和染料加载程序是成功完成全神经节VSD成像的关键步骤。 前者需要去除覆盖水蛭节段神经节神经元的鞘,这是成功染料加载所需的。 后者需要使用一对蠕动泵同时轻柔地将新一代VSD VF2.1(OMe).H流入神经节的两侧。 我们期望所描述的技术广泛地转化为其他薄且相对透明的神经系统中的宽视场VSD成像。

【背景】双面显微镜是一种宽视野荧光成像系统,由一对精确对准的显微镜组成,用于观察来自对面的神经元制剂并且一次显示不同的焦平面(Tomina and Wagenaar,2017)。通过将该光学系统与新一代电压敏感染料(VSD),VoltageFluor(Miller等人,2012; Woodford等人,2015),荧光可以同时从不同深度的神经元捕获编码具有高保真度膜电压的信号。我们将这种泛神经元记录系统应用于药用水蛭的神经系统,我们利用电生理学方法诱发虚构行为并定量控制可识别神经元的膜电位(Tomina and Wagenaar,2017)。在孤立的神经系统中诱发假想行为:局部弯曲是由压力敏感神经元的细胞内刺激引起的,而游泳和爬行则分别通过细胞外刺激分段神经节和尾脑神经引起。我们能够分析水蛭神经系统功能单元中几乎所有单个可识别神经元的动态,使我们能够构建这些神经元在各种行为中扮演的角色的功能图。该成像技术可能应用于其他具有多层体细胞的神经系统,例如Aplysia的蹬神经节 。

对于使用双面显微镜成功进行VSD成像,三个步骤至关重要:(1)解剖目标神经系统,(2)染料加载以及(3)VSD成像本身。这些程序在我们以前的论文(Tomina和Wagenaar,2017)中没有详细解释,也没有在使用相同类型染料的其他研究中进行详细说明(Miller等人,2012年,Moshtagh-Khorasani 2013年,伍德福德等人,2015年,弗拉迪等人,2016年)。

作为解剖过程的一部分,从目标神经节的表面去除鞘是必要的,以确保染料可以到达神经元。为了使制剂保持健康状态,以适当的方式处理制剂并将染料装入细胞适当的时间长度是非常重要的。在VSD成像过程中,神经系统必须严格固定以抑制运动伪影。所有这些步骤对于使用双面显微镜成功完成全神经节VSD成像至关重要。该协议提供了在水蛭的整个神经节中实现宽视场VSD成像的详细程序。

关键字:电压敏感染料成像, 全脑记录, VoltageFluor, 显微切割, 染料加载过程

材料和试剂

  1. 解剖
    1. 显微切割容器,包括:
      1. 35毫米塑料培养皿的盖子(例如,Nunclon Delta,Thermo Fisher Scientific,目录号:153066)
      2. 1.5mm厚的透明PDMS层(Sylgard 184弹性体,0.5mg试剂盒,Dow Corning,目录号:4019862)
      3. 从上面切下一块矩形窗口(1.6×2.6mm)的透明PDMS(直径13mm,厚度0.65mm)盘(Sylgard 184,如上)

    2. 尼龙头针#6(Emil Artl Elephant品牌)
    3. 小针(Fine Science Tools,目录号:26002-10)
    4. 钨丝,直径50μm(加利福尼亚细丝,目录号:MS138)
    5. 人造池塘水(0.1%海洋盐度盐溶液)(例如,海盐,Instant Ocean,目录号:SS15-10)

    6. 水蛭的神经系统 注:我们使用成年药用水蛭Hirudo verbana(2.5-3岁),这是一种神经行为学领域的标准品种,从尼亚加拉水虱(尼亚加拉瀑布,纽约, www.leeches.bi )。 H. medicinalis同样可以接受。将水蛭保存在一个装满人造池塘水的水族箱(250 x 500 x 300毫米)中,在15摄氏度下充气,然后进行12:12光照:黑暗循环。
    7. 粗大的解剖容器,包括:
      1. 外壳(AN-1321,Bud Industries,Digi-Key Electronics,目录号:377-1740-ND)
      2. 小水族馆鹅卵石(例如,Imagitarium迷你白色水族馆碎石,PetCo)
      3. 深色PDMS(Sylgard 170硅酮,0.9kg试剂盒,Dow Corning,目录号:1696157)

  2. 染料加载
    1. 染料加载盘与流出毛细管连接,包括:
      一个。 35毫米塑料陪替氏培养皿的盖子(例如,Nunclon Delta,Thermo Fisher Scientific,Thermo ScientificTM,产品目录号:153066)
      湾毛细玻璃,标准1.2 x 0.68 mm,4“(A-M Systems,目录号:627000)
      C。环氧胶(例如,Slow-cure 30min Epoxy,Bob Smith Industries Inc.)
      d。透明PDMS(Sylgard 184弹性体,0.5公斤套件,道康宁,产品目录号:4019862)

    2. 用于灌注的管(Masterflex C-flex管,Cole-Parmer Instrument,目录号:06424-13)
    3. 音量限制良好:
      一个3毫米厚的PDMS平板,直径25毫米,有一个16毫米直径的孔被切掉。
    4. 1,000μl移液器吸头(例如VWR,目录号:83007-380)
    5. 200μl移液枪头(如VWR,目录号:53508-783)
    6. 10μl移液枪头(例如,Thermo Fisher Scientific,Thermo Scientific,目录号:490014-502)
    7. 70%的乙醇

  3. VoltageFluor解决方案
    1. VF2.1(OMe).H(由加利福尼亚大学伯克利分校的Evan Miller提供,报道于Woodford等人,2015年)
      注意:替代产品可以商业购买:FluoVolt膜电位试剂盒(Thermo Fisher Scientific,Invitrogen TM TM,产品目录号:F10488 )。
    2. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D4540)
    3. Pluronic acid(PowerLoad TM Concentrate 100x,Thermo Fisher Scientific,Invitrogen TM,目录号:P10020)
    4. 水蛭(Hirudo verbana)生理盐水(参见食谱)
      1. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
      2. 氯化钾(KCl)(Sigma-Aldrich,目录号:P3911)
      3. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:223506)
      4. 氯化镁六水合物(MgCl 2•6H 2 O)(Sigma-Aldrich,目录号:M9272)
      5. 葡萄糖(Sigma-Aldrich,目录号:DX0145)
      6. HEPES(Sigma-Aldrich,目录号:H3375)
    5. VF2.1(OMe).H解决方案(请参阅食谱)

  4. VSD成像
    注意:有关构建双面显微镜的详细信息,请参阅Tomina和Wagenaar(2017)。这里我们描述显微镜的使用。
    1. 记录盘,包括:

      1. 一个35毫米培养皿的盖子(如,Nunclon Delta,Thermo Fisher Scientific,产品目录号:153066)
      2. PDMS基材(Sylgard 184弹性体,见上文)
      3. 圆盖玻璃(直径:18毫米,厚度:0.2毫米,Fisher Scientific,Fisherbrand,目录号:12-546)
    2. 医用敷料(Tegaderm透明薄膜敷料,3M,目录号:9505W,例如亚马逊)
    3. 造型粘土(Crayola,任何颜色都很好)
    4. 镜头清洁纸(例如,Tiffen,目录号:EK1546027T,例如,Adorama)
    5. 用于细胞内记录的玻璃毛细管(标准壁1.00×0.75mm,长丝,4“,A-M Systems,目录号:615000)
    6. 用于细胞外记录的吸引电极,包括:
      1. 玻璃毛细管(标准壁1.20×0.68mm,4“,A-M Systems,目录号:627000)
      2. 带Luer的不锈钢钝针(组件供应,目录号:NE-252PL)
      3. 2.5 mm单声道插孔(例如,CUI,Digi-Key Electronics,目录号:MJ-2506)
      4. 5毫升Luer-Lok注射器(BD,目录号:309646)
      5. 银线(200微米直径,加利福尼亚细线,目录号:100183)
      6. 铸造丙烯酸基座,2“x 0.5”x 0.25“
    7. 石油果冻(例如,凡士林)

设备

  1. 解剖
    1. 显微手术刀,5.0毫米刀片(SharpPoint,Surgical specialties,目录号:72-1551)
    2. 用于切割体壁组织的粗剥离剪刀(例如,Heritage,型号:Electricians Scissors 103C)
    3. 用于解剖结缔组织和神经组织的中型解剖剪刀(,例如,Natsume Seisakusho,目录编号:MB-54-1)
    4. 显微切割剪刀:2毫米切割边缘vannas弹簧剪刀(精细科学工具,目录号:15000-03)用于去除水蛭神经节
    5. 细钳( ,Dumont#5 Dumostar,Fine Science Tools,目录号:11295-10)
    6. 粗镊子(如,Peer-Vigor Tweezer Swiss#5,Peer,目录编号:57.0805)
    7. 立体显微镜
      注意:我们使用蔡司Stemi 2000-CS,放大倍率为6.5-50x,放大倍数为W10x / 21,适用于大多数解剖和蔡司SteREO Discovery V8,放大倍数为10x / 23眼镜片和定制聚焦驱动器,放大率为80x (ZEISS,型号:Stemi 2000-CS和SteREO Discovery.V8)。
    8. 带双鹅颈导光管的LED照明灯(Dolan-Jenner Fiber-Lite,型号:Mi-LED-US-DG)
    9. 解剖用冷却板基座:
      1. DC电源(30 V x 10 A DC, ,Yescom,型号:15SDS019-DCP3010D-09)
      2. 珀尔帖模块(TE Technology,型号:HP-127-1.4-2.5-72P)
      3. 0.25“ - 铝铝
      4. 18号不锈钢
      5. 水族馆胶水

  2. 染料加载
    1. 灌注泵头(Easy-Load II头,Masterflex L / S,Cole-Parmer仪器,目录号:EW-77201-60)
    2. 灌注泵驱动器(经济型驱动器,Masterflex L / S,Cole-Parmer仪器,目录号:07554-80)
    3. 微型燕尾阶段(Siskiyou,产品目录号:DT3-100)
    4. 玻璃切割瓷砖(萨特仪器,目录号:CTS)
    5. 移液器100-1,000μl(,例如,VWR,目录号:89079-974)
    6. 移液器20-200μl(,例如,VWR,目录号:89079-970)
    7. 移液器0.5-10μl(,例如,VWR,目录号:89079-960)
    8. Vortexer(例如,VWR,型号:VM-3000,目录号:58816-121)
      注意:此产品已停产。
    9. 离心机(例如,Spectrafuge,Labnet International,型号:C1301-R)
    10. 带双鹅颈导光管的LED照明灯(Dolan-Jenner Fiber-Lite,型号:Mi-LED-US-DG)
    11. 用于照明器的红色滤光片(660nm长通道,0.5“直径,Edmund Optics,目录号:66-045)
    12. 用于在染料加载/记录盘中钻孔的仪器(如,一台车床,如:Jet Tools,型号:BDB-1340A,目录号:321102AK)

  3. 光学成像
    1. 直立式荧光显微镜(奥林巴斯,型号:BX51)
    2. 倒置式荧光显微镜(奥林巴斯,型号:IX51)的荧光灯。
    3. 顶级显微镜(奥林巴斯,型号:XLUMPlanFLN20XW)的水浸20倍物镜
    4. 5倍物镜顶级显微镜(奥林巴斯,型号:MPlan FLN)
    5. 用于底部显微镜的20倍物镜(奥林巴斯,型号:UCPlanFLN)
    6. 振动隔离表(例如,Newport,型号:LW3048B-OPT或VIS3048-SG2-325A)
    7. 蓝色LED(LedEngin,型号:LZ1-10B200)
    8. LED控制器(根据Wagenaar,2012)
    9. CCD相机(Photometrics,型号:QuantEM 512SC)
    10. 细胞内放大器(A-M系统,型号:1600型)
    11. 四通道差分放大器(A-M系统,型号:1700型)
    12. 火焰/布朗微管拉拔器(Sutter仪器,型号:P-97)
    13. 数据采集板(National Instruments,型号:NI USB-6221)

程序

  1. 制作工具
    1. 粗剥离容器:

      1. 将水族箱鹅卵石的容器底部填充到其高度的一半左右。
      2. 将黑色Sylgard(PDMS)倒入距离顶部10毫米的容器中。
        注意:鹅卵石慷慨覆盖至关重要。

      3. 将PDMS在室温或设置在65°C的实验室烤箱中放置过夜。
    2. 显微切割容器:
      1. 将0.65毫米厚的透明Sylgard(PDMS)层放入一个塑料培养皿中,将一层2毫米厚的层放入另一个培养皿中。
      2. 允许PDMS设置(如上)。

      3. 使用显微手术刀在0.65毫米厚的PDMS层上打开一个带有开放窗口(1.6 x 2.6毫米)的圆盘(直径13毫米)。
    3. 染料盘(图1):
      1. 进入塑料培养皿的中心,钻一个直径超过1.2毫米的孔,使毛细管通过使用皮带驱动台式车床进行装配。
      2. 将毛细管玻璃在气体火焰上加热,并将其成形为具有7mm短腿的“L”。将长腿剪成10毫米。

      3. 将L的短腿从外面插入孔中。

      4. 使用环氧胶将毛细管粘在盘子上。
      5. 将毛细管的末端连接到与短毛细管(18 mm)相连的短管上。该短毛细管连接到灌注泵的管路。

      6. 将2毫米厚的PDMS底层倒入培养皿中,让其静置过夜。
      7. 直接从毛细管上方切出2毫米直径的PDMS孔,确认液体可以通过毛细管。


        图1.双面染料加载系统。 :一种。染料加载盘的顶视图。在盘子的中心有一个流出孔,用于将染料加载到神经节的底部表面。该孔与毛细管和管连接以进行循环泵送。孔周围形成的PDMS衬底可以帮助减少VSD解决方案所需的体积。该盘放置在两个铝块上,以便盘,毛细管和管稳定定位。 B.染料加载盘的侧视图。玻璃毛细管插入碟的底部孔用环氧胶(黄色 )稳定。 (为了清洁,可以通过拉出毛细管和管的未粘合部分来分离盘和管。)C.染料加载系统的流出和进口的空间布置。在其中心窗口上带有目标神经节的PDMS圆盘放置在井的中心,使得从底部的流出物到达神经节的底部表面。吸入毛细血管定位在靠近孔边缘的位置,以免碰撞PDMS磁盘。
        顶部流出放置在神经节顶部表面的上方。

    4. 录制菜
      1. 在塑料盘的中心打一个孔(直径14毫米)。
        注意:这可以使用激光切割机,车床或磨机轻松完成。我们发现用手持式钻机实现清洁孔很困难。
      2. 在中心孔的底部放置一个直径为18毫米的圆形盖玻片,在盖玻片边缘周围涂上PDMS以将其粘附到培养皿上,并让其静置过夜。
      3. 将透明的Sylgard(PDMS)倒入皿中至1.5mm的厚度,再次放置过夜。

      4. 从PDMS中切出一个比圆盘底部的孔略大的圆圈。
      5. 从盖玻片上完全去除任何PDMS基片。

  2. 解剖(视频1:粗解剖,视频2:显微解剖)

    视频1

    视频2

    1. 将水蛭放入盛有盐水的塑料容器中,并用冷冻盐水制成冰块,让水蛭冷却10-20分钟,然后解剖。
      注意:当足够麻醉时,水蛭不会显示当未被麻醉的水蛭头部或尾部被钉住时显示的快速反射扭曲式反应。
    2. 将水蛭转移到一个放在冰箱或冰柜里的“粗解剖容器”(见“材料和试剂”)。
    3. 使用昆虫针固定水蛭进行解剖。
      注意:在整个解剖过程中,将容器放置在冷却板上(请参阅'设备'),并使用立体显微镜进行所有操作。
    4. 根据需要切掉组织以隔离水蛭的中枢神经系统(整个神经索或神经节短链)。
      注意:神经系统周围的血窦需要在成像目标周围切除,而不是在任何其他神经节周围。
    5. 将分离的制剂吸入移液管并将其转移到装有盐水的显微切割容器中(参见“材料和试剂”)。
    6. 将这个容器放在冷却板上,并使用高倍率立体显微镜进行其余步骤。

    7. 将透明PDMS(请参阅'材料和试剂')放在容器中央
    8. 将目标神经节置于盘上,腹侧朝上,在窗口中央居中,以便PDMS基底不接触神经节的背侧。

    9. 使用短小的钨丝将血窦组织周围的神经根周围的血窦组织
    10. 使用微型剪刀或显微手术刀,仔细清洁地从神经节的腹侧和背侧表面取下鞘,步骤如下:
      1. 去除腹侧鞘(图2A)。
      2. 根据需要小心取下神经节,将其倒置在PDMS磁盘的窗口上,注意不要损伤该过程中的任何神经元。
      3. 去除背鞘(图2B)。
      注意(非常重要):在整个过程中,通过使用塑料移液管每2-3分钟用新鲜冷盐水代替盘中的盐水直至准备好染料加载,保持神经节冷却。 desheathing之后的时间至关重要。
      应在15-20分钟内完成脱鞘

      图2.去鞘神经节对于电压敏感的染料加载,覆盖目标神经节的鞘组织(例如,第10节节)从腹侧(A)和背(B)侧。 'R'表示神经节的右侧,即,当动物右侧朝上时,右侧。比例尺= 100μm。

  3. 染料加载
    1. 准备2毫升VSD溶液(800 nM VF2.1(OMe).H)在含1%普鲁兰尼克酸的水蛭生理盐水中,参见食谱),并在室温下在黑暗中保持至少1小时。
      注意:使用新鲜制作的冷VSD溶液会导致明显的染色不良,从而导致信号质量急剧下降。确保在昏暗的红光下进行染料加载以避免漂白染料。 
    2. 将'容积限制孔'放入'染料加载皿'(参见'材料和试剂'),集中在底部流出毛细管上。

    3. 将顶部流出毛细管松散地放在井的中心。
    4. 用VSD溶液填充井,将两个进水毛细管浸入靠近井边的溶液中。
    5. 将两台蠕动泵的流速设定为约1.1 ml / min(速度旋钮2级),并确认灌注效果良好。
      注意:确保流出毛细管内没有气泡积聚。如果气泡卡在毛细血管内,则暂时增加抽气速度以喷出气泡。在启动VSD染色之前必须完成去除气泡。
    6. 将带有仍附着在其上的神经节的PDMS盘转移到染料加样皿中的孔中,并使用一个或两个昆虫针将其固定在悬挂在流出毛细管上的神经节上(图1A)。
      注意:此时神经节在盘下方'悬挂',背侧朝下。
    7. 使顶部流出毛细管接近神经节(大约1mm距离;图1C),并使用两个蠕动泵使VSD溶液(1.1ml / min流速)循环8分钟。
    8. 翻转PDMS盘,将倒置的神经节倒置在井内(所以神经节位于盘顶部,背面朝上),注意保持它在底部出流毛细管的中心。
    9. 使用两个蠕动泵将VSD解决方案再循环12分钟。
      注意:为了在成像准备的两侧获得均衡的亮度,准备的底部必须比顶部染色更强烈,因为顶部聚焦平面上的激发光要比顶部聚焦平面上的激发光更亮底部重点平面。顶部流出比底部流出更有效地染色神经节,因此步骤C7中8分钟循环的选择和步骤C9中12分钟的选择导致背侧比腹侧更强烈地染色。这是通过设计:它在成像过程中有效地补偿神经节底部的激励减少。
    10. 染料加载后用冷盐水轻轻洗涤制剂,并用普通冷盐水完全代替含有VSD的盐水。
    11. 用70%乙醇和蒸馏水循环清洗流出管和毛细管。
      注意:这可以在成像后完成。

  4. 光学成像
    1. 通过将围绕神经根的血窦组织牢固地固定到PDMS盘并通过将相邻的连接物夹在医用敷料的小片之间(图3C)来固定目标神经节以用于成像,以将任何运动伪影最小化。

    2. 在'记录盘'的盖玻片边缘放置少量凡士林(见“材料和试剂”;图3A)。
    3. 将圆盘与神经节放在记录盘的中心,将其向下推入凡士林。
    4. 将神经节周围相邻结缔组织周围的血窦组织拉出,以便组织可以紧紧地固定在记录盘的周边PDMS基底上(图3B)。

    5. 在成像之前用镜头清洁纸清洁防护玻璃的底部表面

      图3.用于双面VSD成像的记录盘A.记录盘( top )中心区域的顶视图和正面剖视图( >底)。在将含有制剂的PDMS盘放入盘中之前,将凡士林应用于盖玻片的周边。 B.在盖玻片( top )上设置一个准备工作的PDMS磁盘俯视图和正面剖视图( bottom )。为了最小化运动伪影,通过将相邻神经节周围的血窦组织向下固定在盘的外部区域的PDMS基底上来产生张力。 C.夹在两块医用敷料( top )和正面剖视图( bottom )之间的相邻结缔组织。这些医用敷料被固定在PDMS磁盘上。

    6. 将记录盘放在双面显微镜的台上(图4A和4B)。
    7. 在红光曝光下使用顶部(直立)显微镜调整准备的方向。
    8. 通过将电极放置在目标神经或细胞体附近准备细胞外和/或细胞内记录(图4C)。
      注:染料加载结束和VSD成像开始之间的时间间隔通常为20-25分钟。在这里花费更多时间可能会对神经节的健康产生负面影响。


      图4.双面显微镜。 :一种。双面显微镜的概述。奥林巴斯BX立式显微镜的荧光序列在Olympus IX倒置显微镜的主体顶部安装有定制对焦架。图像由两个CCD相机获取。 B.精确对准双侧VSD成像的顶部和底部20x物镜。记录盘放在塑料台的切口( white )中。粘性粘土用于固定盘子的位置。 C.同时电生理学和双侧VSD成像。细胞内和细胞外电极都可以接触到制剂的正面。

    9. 将目标神经吸入吸入电极和/或刺入靶细胞。

    10. 使用顶部和底部的物镜将神经节的顶部和底部表面粗糙地聚焦
    11. 使用LED控制器的强度控制或显微镜的光圈,调整图像的亮度以获得最佳亮度,但不要使CCD相机饱和。
    12. 确定顶部和底部成像的最终焦点。
      注意(重要):在所有这些准备工作中,经常用冷盐水代替盐水。
    13. 使用VScope开始VSD成像/电生理学试验(Wagenaar,2017)。
      注意:确保在黑暗环境下进行光学成像。
    14. 完成VSD成像实验后,在不同的聚焦深度获取神经节顶部和底部的快照,以便以后创建神经节的“聚焦堆叠”图像(图5)。这些将用于绘制VScope中的感兴趣区域(ROI)。


      图5.神经节的可视化 A.顶级相机拍摄的第10节神经节腹侧的单一快照图像。 B.用自适应对比度增强(ACE)滤波器处理后的相同图像。 C.由不同深度的腹部方面的多个快照构建的焦点堆叠图像。 D.用ACE过滤器处理后的相同图像。比例尺= 100μm。

数据分析

  1. 即使在正在进行的实验中,VScope(Wagenaar,2017)也可用于检查获取的光学信号是否显示神经元活动(自发活动,刺激诱导反应或虚构行为)。其余分析通常在实验结束后进行。
  2. 使用VScope的“自适应对比度增强”滤镜(图5)在焦点堆叠的图像中绘制ROI,然后将ROI信息传输到单独的试验中。
  3. 使用VScope提供的功能将数据加载到Octave中。

  4. 使用运动校正和去漂白算法清理VSD成像数据(Tomina and Wagenaar,2017)。
  5. 使用Octave进行与您的实验相关的进一步统计分析,例如(图6)。
  6. 检查细胞内记录是否与选定神经元的VSD信号相匹配,以确认光学记录的可靠性。同时检查在代表性神经元如腹侧/背侧运动神经元(DI1,VI2,DE3和VE4)中是否观察到定型运动模式,特别是当实验涉及假想行为时(图6)。


    图6.双面VSD成像。 A和B两个CCD表面图像(A中的腹侧; B的背侧)同时用两个CCD相机捕获。比例尺= 100μm。 C.虚拟游泳期间选择的电生理和VSD踪迹:来自后段神经根的细胞外记录(第13节神经节的DP神经),显示节律性背侧兴奋性运动神经元爆发;来自左AE细胞(运动神经元,用*标记)的细胞内记录和同时光信号显示匹配的膜电位振荡;来自腹面的VSD信号:双侧AE细胞和Retzius细胞(神经调节神经元);来自背侧表面的VSD信号:背侧和腹侧抑制性和兴奋性运动神经元DI-1,VI-2,DE-3和VE-4。比例尺=时间为1秒,膜电位为10 mV,荧光信号为0.2%ΔF/ F。

注意:示例数据,代码和详细应用手册可在Dryad Digital Repository中找到( https://doi.org/10.5061/dryad.m20kh )。

笔记

  1. 应该快速有效地进行解剖(典型的10-30分钟用于隔离水蛭神经系统,15-20分钟用于双面脱鞘),并且盐水应该经常用冷的生理盐水代替(每2-3分钟)用吸管,因为水蛭神经系统的健康对实验的成功至关重要。
  2. 为了抑制来自神经索周围鞘内收缩组织的运动伪影,在染料加载后,通过将剩余的血窦固定在PDMS基底上,应该物理拉伸结缔组织和外侧神经。如果在成像过程中观察到不可忽略的运动伪影,则应增加其拉伸强度。
  3. 溶于DMSO中的VF2.1(OMe).H储备液应保存在4°C的冰箱中。为保持染色质量,请勿将染料置于室温或深度冷冻室中。
    注意:根据VoltageFluor(FluoVolt Membrane Potential Kit)商业产品的使用手册,该产品应存放在2-8°C。
  4. 我们定性地评估了我们的标本如何使用实验期间获得的荧光图像和信号进行染色(实例如图5和图6所示)。这是目前确认染色过程进行得顺利的唯一方法。
    在染料加载过程中寻找神经节的视觉变化并不能提供关于染色质量的可靠信息 注意:我们建议初学者在水蛭中进行VSD成像,首先通过针对Retizus细胞或任何其他易受刺激的细胞,在细胞内记录和VSD信号之间获得一致的结果。

食谱

  1. Hirudo verbana 生理盐水

    注意:用NaOH或HCl调节pH值至7.4。
  2. 800nM VF2.1(OMe).H溶液(1ml)
    1. 将8μl100μMVF2.1(OMe).H(储存在DMSO中的溶液,保存在冰箱中)分装在微管中并预先保存在冰箱中
    2. 将其与10μlpluronic acid
      混合
    3. 加入蚂le盐水使其最终体积为1毫升,并将其涡旋
    注:此配方适用于市售的VoltageFluor染料。

致谢

我们感谢Evan Miller提供VF2.1(OMe).H染料; Annette Stowasser在开发双面显微镜原型和许多有帮助的对话中的角色; Angela Bruno就数据分析进行有益的讨论;吴彩塘对稿件进行阅读和检查。这项工作得到了Burroughs的欢迎基金的支持,通过科学界的职业奖和国家神经疾病与中风研究所,通过拨款R01 NS094403(都给DAW)。 YT得到了JSPS海外研究奖学金的支持。该协议是根据Tomina和Wagenaar(2017年)发布的程序改编的。本作品的作者声明不存在利益冲突。

参考

  1. Frady,E.P.,Kapoor,A.,Horvitz,E。和Kristan,W.B.,Jr.(2016)。 可扩展的半监督功能性神经造影揭示了行为网络中的典型神经元 Neural Comput 28(8):1453-1497。
  2. Miller,E.W.,Lin,J.Y.,Frady,E.P.,Steinbach,P.A.,Kristan,W.B.,Jr.和Tsien,R.Y。(2012)。 通过分子导线通过光致电子转移来光学监测神经元中的电压 Proc Natl Acad Sci USA 109(6):2114-2119。
  3. Moshtagh-Khorasani,M.,Miller,E.W。和Torre,V。(2013)。 水蛭神经节中神经元的自发电活动 Physiol Rep 1 (5):e00089。
  4. Tomina,Y.和Wagenaar,D.A。(2017)。 双侧显微镜可以实现药用水蛭膜电位的全神经节显像。 a> Elife 6。
  5. Wagenaar,D.A。(2012)。 光学稳定的快速切换发光二极管作为功能神经影像的光源。 PLoS One 7(1):e29822。
  6. Wagenaar,D.A。(2017)。 VScope - 使用多个摄像头和电生理学进行电压敏感染料成像的数据采集和分析。 a> 开放研究软件杂志 5:23。
  7. Woodford,CR,Frady,EP,Smith,RS,Morey,B.,Canzi,G.,Palida,SF,Araneda,RC,Kristan,WB,Jr.,Kubiak,CP,Miller,EW和Tsien,RY )。 改进的用于光学感应神经元电压的PeT分子 J Am Chem Soc 137(5):1817-1824。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Tomina and Wagenaar. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tomina, Y. and Wagenaar, D. A. (2018). Dual-sided Voltage-sensitive Dye Imaging of Leech Ganglia. Bio-protocol 8(5): e2751. DOI: 10.21769/BioProtoc.2751.
  2. Tomina, Y. and Wagenaar, D. A. (2017). A double-sided microscope to realize whole-ganglion imaging of membrane potential in the medicinal leech. Elife 6.
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