Analysis and Quantification of Functional Regeneration of Dendrite and Axon of PVD Neuron After Laser Injury in Caenorhabditis elegans

Materials and reagents

Biological materials

1. C. elegans strains (wdIs52)(F49H12.4::GFP) expressing GFP in the PVD neuron. The C. elegans strains utilized in this work were obtained from the Caenorhabditis Genetic Center (CGC) in Minneapolis, USA. Please refer to wormbook.com for fundamental worm culture procedures

2. OP50 E. Coli bacteria obtained from CGC USA

Reagents

1. Sodium chloride (NaCl) (Sigma, catalog number: 71376)

2. Tryptone (TM Media, catalog number: 3514)

3. Peptone (TM Media, catalog number: 1506)

4. Agarose (Sigma, catalog number: A9539)

5. Agar agar type 1 (TM Media, catalog number: 242M)

6. Cholesterol (Sigma, catalog number: C8667)

7. Levamisole hydrochloride (Sigma, catalog number: L0380000)

8. Calcium chloride (CaCl₂) (Qualigens Fine Chemicals, catalog number: 22205)

9. Magnesium sulphate heptahydrate (MgSO₄.7H₂O) (Merck, catalog number: 193645)

10. Potassium phosphate monobasic anhydrous (KH₂PO₄) (Merck, catalog number: 193205)

11. Absolute ethanol (Merck, catalog number: 100983)

12. Sodium phosphate dibasic anhydrous (Na₂HPO₄) (Merck, catalog number: 193609)

13. Ammonium Chloride (NH₄Cl) (Merck, catalog number: 193621)

Solutions

1. Nematode growth medium (NGM) plates (see Recipes)

2. B broth (see Recipes)

3. OP50 culture (see Recipes)

4. 5% agarose solution (see Recipes)

5. M9 buffer (see Recipes)

Recipes

1. Nematode growth medium (NGM) plates

For preparation of 1 L of media, add 3 g of NaCl, 2.5 g of peptone, and 20 g of agar to 700 mL of MilliQ water; then, bring up the volume to 1,000 mL. Autoclave the media, let it cool down to 55 °C, and add the following solutions after filter sterilization using a 0.22 μm filter: 1 mL of cholesterol (10 mg/mL prepared in absolute ethanol), 1 mL of 1 M CaCl₂, 1 mL of 1 M MgSO₄.7H₂O, and 25 mL of 1 M KH₂PO₄ under sterile conditions. Mix well and pour media into 60 mm Petri plates with 10 mL of media per plate. Keep at room temperature for 24 h and then store at 4 °C for later use.

2. B broth

Add 0.5 g of tryptone and 0.25 g of NaCl to 50 mL of MilliQ water. Autoclave and store it at 4 °C.

3. OP50 culture and seeded NGM plates

Thaw the OP50 bacteria from its glycerol stock by streaking it onto an NGM plate. Incubate this plate at 37 °C for 12 h. Using a sterile tip, pick one colony of OP50 bacteria from the freshly streaked plate and place it into autoclaved B broth. Incubate it overnight at 37 °C. Use this liquid culture for seeding the NGM plates for worm growth and maintenance. Parafilm the freshly streaked plate of OP50 and the liquid culture in B broth and store in a 4 °C refrigerator. This liquid culture can be used for up to 2 weeks.

From the cultured media, pour around 5 mL in a sterile 60 mm plate. Take a sterile glass L-shaped rod, dip in the culture of OP50 in the 60 mm plate, and use this L rod to spread the culture in a square patch on the sterile NGM plate. Sterilize the L rod over a flame in between spreading on multiple plates. Incubate these plates at 37 °C for 10–12 h so that a thin square-shaped patch of OP50 bacteria grows. These plates are used for regular worm maintenance.

To create a uniform lawn for behavioral experiments, pour around 100 μL of cultured OP50 on the sterile NGM plates and spread the culture onto the plate by swirling the Petri plate. Do not put the lids on until the culture has dried completely. Incubate these plates at 37 °C for 10–12 h so that a uniform circular lawn appears on the plates. These seeded plates can be stored at 15 °C for a week.

4. 5% agarose solution

Weigh 0.15 g of agarose in a test tube. Add 3 mL of 1× M9 buffer to it. Hold the test tube with the test tube holder and heat it up on a Bunsen burner or spirit lamp until the solution boils. Keep that test tube in the heating block set to 60 °C for 5 min to remove bubbles. When bubbles in the solution are cleared up, use it for making slides. Keep that test tube in the heating block until the experiment is done.

Note: Make sure to use a solution clear of bubbles as they may hinder imaging. Make fresh agarose solution every time imaging or injury experiments are to be conducted.

5. M9 buffer

For preparing 5× M9 buffer, add the following salts to make their final concentration as mentioned: 0.1 M KH₂PO₄, 0.2 M Na₂HPO₄, 0.04 M NaCl, and 0.1 M NH₄Cl. Mix the salts (heat to 60–65 °C if needed). Autoclave the solution and store it at room temperature. Dilute the solution to 1× using MilliQ water for agarose preparation.

Laboratory supplies

1. Test tube, 5 mL (Praveen Scientific Corporation, catalog number: 40487)

2. Tips [Tarsons, catalog numbers: 521000 (10 μL), 521010 (200 μL), 521016 (1,000 μL)]

3. Filter tips, 200 μL (Tarsons, catalog number: 527104)

4. Micropipette [Gilson, catalog numbers: F144054M (P2), F144056M (P20), F144058M (P200), F144059M (P1000)]

5. Disposable sterile Petri dishes, 60 mm (Praveen Scientific Corporation, catalog number: 20440)

6. Glass slides (Blue Star, PIC 1.75 mm long × 25 mm wide, 1.35 mm thick)

7. Coverslips (Blue star, 0.13 mm thick, 18 mm square)

8. Thin glass capillary (World Precision Instruments, catalog number: TW150-6)

9. Rubber tube (7 mm outer diameter, 4 mm inner diameter)

10. Butterfly scalp vein set (HMD, 22G × 19 mm)

11. Erlenmeyer flask (Borosil Scientific)

12. Measuring cylinder (Borosil Scientific)

13. Pasteur pipette (VWR, catalog number: 612-1798)

14. Spatula

15. Rubber bulb

16. Permanent marker

17. Worm pick of platinum wire with 99.99% purity of 0.15 mm diameter for the harsh-touch stimuli and 0.3 mm diameter for regular worm picking

Equipment

1. Weighing balance [Sartorius, model: BSA224S-CW, 220 g(max), d-0.1 mg]

2. Laminar flow hood (Atlantis)

3. Luer connector (isolated from the butterfly scalp vein set)

4. Stereomicroscope (Nikon, model: SMZ745, zoom 5× max)

5. Leica stereomicroscope with attached camera for taking videos and still images of the worms over the NGM plate (Leica, model: M165FC), zoom 12× max, attached camera (Leica, model: MC120HD), resolution 2.5 megapixels (1,216 × 912), pixel size 3.34 μm, field of view 15 mm × 15 mm required for harsh touch videos

6. Two-photon microscope (Bruker ULTIMA multiphoton microscopy system) coupled with two spectra-physics, tunable infra-red femtosecond lasers (λ = 690–1,040 nm) having automated dispersion compensation (MaiTai with DeepSee), Conoptics Pockel cells with microseconds temporal resolution to control the output of the laser, two independent X-Y scan galvanometer mirrors for simultaneous imaging and cutting (6 mm scan galvanometer for imaging, 3 mm scan galvanometer for cutting). Imaging laser set at 920 nm with 50 fps (frames per second) pulse width, cutting laser set at 720 nm with 80 fps pulse width, irradiation time 20–30 ms, laser power 23 mW, lateral point spread function (PSF) ~400 nm, Z axis PSF ~1.5 μm, 60× Olympus water immersion objective (60×, 0.9 NAP). The IR femtosecond lasers can be switched on from MaiTai SpectraPhysics software (Figure 1E) or from the Prairie View software itself. The resting wavelength at which the lasers are switched on or off is 820 nm. Pockel-1 is used as a controller for primary laser power (used for imaging) (Figure 1E), which is present in the main home tab of Prairie View software. The output signal is detected by the photomultiplier tube (PMT) detector unit (green and red). The cutting laser is controlled by Pockel-2 (Figure 1E). The cutting laser controls reside in the electrophysiology tab in Prairie View software, where the power of the cutting laser and pulse duration can be tweaked

7. Confocal microscope (60×/1.4 NA oil objective of Nikon A1R confocal system for GFP imaging: 488 nm laser; for RFP imaging: 543 nm laser; intensity 4.0 and gain 48.3; pinhole set 1.0 AU; using AX galvanometer scanner with up to 8,192 × 8,192 pixels, 10 fps speed, 512 × 512 resolution image acquisition, dwell time 2 ms, averaging 2 frames, frame time 7.0 s, FITC scan mode, NPARC detector unit for imaging, Z-stack image acquisition at range of 25 μm, slice thickness 1 μm)

8. Worm incubator at 20 °C

9. Bacterial incubator at 37 °C

10. Worm pick platinum wire

11. Eyelash pick

12. Heating block kept at 60 °C (Neolab, model: TC544)

Software and datasets

1. Leica microscope software LAS V4.12

2. Prairie View software for two-photon microscope v5.4

3. MaiTai Spectra-Physics software

4. Nikon imaging software (NIS) Elements-C (imaging software)

5. ImageJ FIJI (analysis software)

6. GraphPad Prism v10.2.3

Procedure