Microinjection

Microinjection of membrane-impermeant, nontoxic tracers has been the first technique used to identify and map the communication network of a wide variety of cell systems. The approach is still widely used to study cell coupling in cultures (see Fig. 1) and is practically the only technique available to extend such studies to intact tissues (17). Because it allows for a selective loading of tracers, microinjection permits correlation of morphological and functional data from individual cells. Furthermore, because the onset and duration of the tracer injection can be accurately controlled, the technique is also instrumental in kinetic studies aimed at evaluating the rate of transfer of a tracer from one cell to another. The major limitation of microinjection is that it requires some special equipment and skill to prevent damaging the cells, or at least to detect such damage during the injection. Another limitation is that only a few cells may be microinjected, usually not at the same time. Thus, the technique is not convenient when a large number of cells need to be simultaneously monitored for intercellular communication.

Fig. 1. Microinjection of a gap junction-permeant tracer. (A) Lucifer Yellow was introduced via a microelectrode (e) into a neonatal rat cardiomyocyte. Within seconds, the tracer diffused from the injected cell into several neighboring companion cells. Note, however, that adjacent fibroblasts did not receive the tracer. (B) The experiment was repeated, within the same culture dish, 15 min after addition to the culture medium of 1 mM heptanol, a blocker of connexin channels. As a result of this blockage, the intercellular exchange of Lucifer Yellow was drastically reduced. (C,D) Phase-contrast views of the fields shown in A and B, respectively. Bar, 20 ^m.

Fig. 1. Microinjection of a gap junction-permeant tracer. (A) Lucifer Yellow was introduced via a microelectrode (e) into a neonatal rat cardiomyocyte. Within seconds, the tracer diffused from the injected cell into several neighboring companion cells. Note, however, that adjacent fibroblasts did not receive the tracer. (B) The experiment was repeated, within the same culture dish, 15 min after addition to the culture medium of 1 mM heptanol, a blocker of connexin channels. As a result of this blockage, the intercellular exchange of Lucifer Yellow was drastically reduced. (C,D) Phase-contrast views of the fields shown in A and B, respectively. Bar, 20 ^m.

3.1.1.1. Preparing the Cells to Be Injected

A preliminary condition for a successful microinjection is that the cell under study should not move during impalement. This is easily achieved with cells in monolayer culture that are grown on a dish suitable for the injection setup, and thus require no further preparation to be injected. The problem may be more complex for immobilizing on an adequate support tridimensional cell assemblies, such as intact tissues. To this end, we routinely use standard 35-mm culture dishes coated with Sylgard and polylysine (for alternatives, see Note 6).

1. Make Sylgard-coated dishes:

Prepare 184 Sylgard silicone elastomer (Dow Corning) as directed by the manufacturer.

Plate 0.5 mL of Sylgard per 35-mm tissue culture dish.

Store at room temperature.

2. Make a poly-L-lysine solution:

Dissolve 1-10 mg of poly-L-lysine hydrobromide (mol wt 150,000-300,000;

Sigma Chemical) in 10 mL of phosphate-buffered saline (PBS), pH 7.2. Put in each dish 2 mL of polylysine solution. Incubate for 1 h at room temperature. Rinse 3x for 5 min each in PBS.

3. Just before the microinjection:

Position a tissue fragment in the center of a Sylgard and poly-L-lysine-coated dish, within a 20-^L drop of albumin- and serum-free medium. After 5-30 min (a longer time period should be provided for attachment of small pieces) at room temperature and within a humidified chamber, remove the 20 ^L of medium. Slowly add the medium to be used during the injection experiment. Cover the medium with a thin layer of light liquid paraffin (BDH Laboratory

Supplies, Poole, UK) to avoid evaporation. Transfer the dish on the heated (37┬░C) stage of the injection microscope.

3.1.1.2. Preparing the Electrodes

Electrodes are pulled from filament-containing borosilicate glass capillary tubing (1.2 mm external diameter, World Precision Instruments ref. TW120F-4) using a micropipet puller that is adjusted to give the desired electrode resistance. Electrodes of 50-60 MQ when filled with 3 M KCl are adequate for most uses (when filled with solutions of tracers, these electrodes show resistances 4-20x higher, depending on the solvent used). A few hours before the experiment:

1. Pull the electrodes using settings adapted to the type of electrode you need and puller equipment you use.

2. Bend slightly the electrode shaft on a small gas flame.

3. Fill only the tip of the electrode with a stock tracer solution using a 34-gauge nonmetallic syringe needle (MicroFil; WPI) fitted on a 0.22-^m pore filter (Sterile Acrodisk®13, cat. no. 4454, Gelman Sciences).

4. Store the filled electrodes in a clean, humidified container.

6. Just before each injection, fill the shaft of the electrodes with the injection solution, using a 34-gauge nonmetallic syringe needle fitted on a 0.22-^m pore filter.

3.1.1.3. Preparing the Electrode on its Holder

The conducting silver wire of the electrode holder should be coated as follows.

1. Prepare the following coating solution: KCl 7.456 g

Store at room temperature.

2. Put 10 mL of coating solution in a beaker.

3. Place in it the silver wire to be coated.

4. Connect the end of the silver wire that will link the electrode to the injection setup to the positive pole of 4.5 V battery (the negative pole of the battery is also linked to another silver wire that is immersed in the coating solution).

5. Check for the development of gas bubbles along the wire connected to the negative pole of the battery.

6. Disconnect the battery after 5 min, when the silver wire of electrode holder has darkened.

7. Mount the chlorinated wire on the electrode holder.

8. Place the tracer filled electrode in the holder.

9. Connect the electrode holder to the head stage of the micromanipulator.

3.1.1.4. Penetrating a Cell

1. Under dark-field or phase-contrast illumination, position the electrode over the area (in tissues) or the perinuclear region of the cell (in monolayer cultures) to be injected, using a 6.3x objective.

2. Shift on the 25x or 40x objective lens.

3. Slowly lower the electrode against the cell membrane.

4. Compensate for series resistances using the balance control of the amplifier.

5. Compensate for electrode resistance using the neutral control of the amplifier.

6. Penetrate individual cells by briefly "vibrating" the electrode, using the negative capacitance control of an amplifier (this procedure also results in the ejection of some dye).

7. Check for correct penetration by a drop of resting membrane potential (for all tracers dissolved in a salt solution) and rapid filling of the injected cell (if the selected tracer is fluorescent).

8. As soon as the adequacy of the penetration has been ascertained, turn off the UV illumination to prevent photoabsorption which could damage the cell (if a video recording is made, the UV light should be decreased as much as possible with inert absorption filters and the signal amplified with an adequate high sensitivity camera) to minimize the binding of tracers to cytoplasmic and nuclear components.

9. Start injecting the tracer.

3.1.1.5. Injecting a Tracer

1. Apply to the electrode hyperpolarizing (negative) or depolarizing (positive) current pulses, depending on whether the selected tracer has a net negative or a positive charge. The duration, amplitude, and frequency of these pulses can be varied depending on the type of electrode used (the amount of current that can be injected decreases per unit time with increasing electrode resistance), the amount of tracer to be injected, and the cell type. We routinely use square, 0.1-nA pulses of 900 ms duration and 0.5 Hz frequency for 3-5 min.

2. During the injection, check for maintenance of adequate penetration by the persistence of a stable resting membrane and, in the case of fluorescent tracers, by the persistence of a high cell fluorescence.

3. At the end of the experiment, interrupt the current pulses.

4. Gently pull the the electrode out of the cell.

3.1.1.6. Recording the Injection

1. If you use a photographic camera, record each injection site before and/or after pulling out the injection electrode, using Kodak Ektachrome 400 daylight film for color slides and a constant exposure time of about 20 s.

2. If you use a video camera, record each injection online, using a high-sensitivity, intensifying equipment (e.g., TM-560, Pulnix; http://www.pulnix.com) and a VO-5630 U-Matic recorder (Sony; http://www.sony.com).

3.1.1.7. Analyzing the Injection

If a fluorescent tracer was chosen, the injected cell and its coupled neighbors may be immediately visualized under a microscope before (in the case of tracers that do not have affinity for cell components) or after fixation of the specimens (in the case of tracers that bind to cell components). If Lucifer Yellow or neurobiotin is chosen, it is further possible to identify the cells containing the tracer (see Note 7), after processing of the tissues for light and electron microscopy (9,10,17,24,26).

3.1.1.7.1. For Light Microscopy

1. Fix the injected sample 30-90 min at room temperature in a 4% solution of paraformaldehyde.

2. Rinse 3x for 10 min in PBS.

3. Dehydrate by 10-min passages in a sequence of 30%, 50%, 70%, and 95% etha-nol solutions.

4. Dehydrate by two 20-min passages in absolute ethanol.

5. Soak the specimens twice for 15 min in propylene oxide (absolute ethanol should be used if the preparation was grown in a plastic dish).

6. Infiltrate the specimen for 2 h in a 1:1 (v/v) mixture of Epon 812 and either propylene oxide or absolute ethanol.

7. Infiltrate the specimen for 12 h with pure Epon.

8. Embed the specimen in freshly prepared Epon 812.

10. Cut serial sections of 1-5 ^m thickness.

11. Screen the sections under a fluorescence microscope for injected and coupled cells which may be recognized by a persistent fluorescence labeling.

3.1.1.7.2. For Electron Microscopy

1. Fix the injected preparation 90 min at room temperature in the 4% paraformalde-hyde solution, supplemented with 0.1-1% glutaraldehyde.

2. Proceed through steps 2-9 as indicated for light microscopy in the previous section.

3. Cut section of about 600 nm thickness.

4. Screen sections under an electron microscope.

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