In vitro reconstruction of neuronal loops: experiments on methods for building a simple model system

Summary

We can reconstruct the S-cell network that initiates the basic respiratory behavior on cultured cells of the freshwater mollusc phylum Cnidaria by using in vitro cell culture techniques. This in vitro reconstructed network is capable of generating rhythmic forms of locomotion similar to those seen in the whole. Thus, this in vitro cell culture method allows us to perform fundamental studies on the neuronal basis of rhythmic activity that are difficult to achieve in either whole or incomplete animals.

Operation method

Simple modeling experiments for reconstructing neuronal loops in vitro

Move

makings

move Aseptic environment, necessary for cell culture

The majority of cell culture processes need to be carried out in ultra-clean benches. With proper sterilization, airborne microorganisms can be prevented from contaminating the culture medium.

Bench Sterilization

The following procedure is strictly followed before each experiment until the bench is closed.

1. Turn on the bench blower to produce airflow.

2. Spray all surfaces in the workbench with 70% ethanol.

3. Wet a paper towel with ethanol; wipe all surfaces in the bench (ethanol sprayed from the bottle can damage the filter).

4. Wait for bench to dry, spray again if necessary.

NOTE: Spray your hands with ethanol from this step until you work inside the bench.

5.70% Ethanol soak all instruments to be used (forceps, scissors, dissecting trays, etc.) and leave them in the workbench to dry.

6. Since all solutions used in cell culture are sterile (including antibiotic saline, special media, conditioned media, lmol/L dextrose), all bottles should be opened only in a properly sterilized bench.

Take care to prevent accidental contamination

Do not eat or drink near the bench (especially drinks and food made from yeast).

Keep the workbench tidy.

Spray any items that have been temporarily taken out of the bench with ethanol before putting them back in (e.g. tweezers) and spray your hands with ethanol before working inside the bench, but do not allow ethanol to get into the petri dishes.

Anatomy of the vertebrate solid snail

Shelled conchs were immersed in 10%-25% Listerine disinfectant for lOmin, after which they were pinned to a dissecting tray containing ABS solution and the CNS was removed in a sterile environment (Syedetal.1990;Ridgwayetal.1991).

Aspiration of isolated cells

To prevent neurons from adhering to the glass wall of the pipette, the inside of the pipette should be coated with a layer of Sigmacote or serum.Sigmacote (Sigma catalog #SL-2, a special cinnamon-heptyl-burning solution) creates a strong, fine microfilm on the glass that prevents cells from adhering to the pipette.

Step one.

1. Use a capillary glass tube with a diameter of 1.5 mm, taking care that there are no burrs, filaments, etc..

2. In a fume hood, suck the Sigmacote into the capillary glass tube (by capillary siphonage) and invert it so that the Sigmacote flows out of the other end. Repeat the procedure several times and leave it to dry overnight. Be careful not to manipulate any toxic chemicals in an ultra-clean bench used for tissue culture, as this can be a serious health hazard!

Step 2

Prepare cell separation tubes using Sigmacote-treated capillary glass tubes.

3. Pull the capillary glass tubes on the Electrode Puller as you would for a pointed intracellular electrode. This will result in electrodes with long and good tips.

4. Cut off half of the long tip of the electrode with a diamond knife pen. The length of the cut-off will determine the final diameter of the cell separation pipette.

5. Apply slight pressure at the tip to break the tip. The electrode will break cleanly.

6. Polish the tip of the electrode over a microfurnace flame. This removes some of the sharp edges which are harmful to neuronal survival. Determine the diameter of the tip using the ruler on the microfurnace. It is important to note that the inner diameter of the electrode tip is always slightly larger than the cell body to be isolated.

7. Remove the electrode from the micromelting furnace flame and polish the opening at its upper end (opposite end from the tip) over the flame of the Bunsen burner to prevent the electrode from damaging the pipette's tubing and clogging the pipette tip.

Cell separation

Neuronal isolation refers to the process of separating individual cell bodies from the central nervous system and placing them in a suitable culture environment. It is a very precise and highly complex process that can be quite difficult for beginners. With practice, patience, persistence and perseverance, isolating a single specific neuron becomes relatively easy. The basic steps of the operation are described below.

Note: All steps should be performed aseptically in an ultra-clean bench.

1. Place an appropriate number of central circumflex ganglia in ABS-containing falcon3001 plastic petri dishes and subsequently transfer them between 3 falcon dishes, each containing 3 ml of ABS, for 10-15 min in each dish. when transferring between dishes, use a deterrent to hold the connective tissue attached to the ganglion, taking care not to damage the ganglion. To avoid cross-infection between petri dishes, cover them at all times except when transferring ganglia.

2. During treatment with antibiotics, prepare 2 falcon dishes for enzyme treatment. Add 3 ml of DM to each petri dish, followed by 6 mg of trypsin or 6 mg of trypsin inhibitor, respectively (i.e., 2 mg/ml at a final volumetric concentration of 0.2%). Mark the petri dishes appropriately to avoid confusion or other possible errors.

3. After rinsing, the brain tissue blocks (12/dish) were transferred to Petri dishes containing trypsin and DM and left at room temperature (18~2undefinedC) for 20~25 min. Shaking every 5 min ensured that the connective tissues around the ganglion were uniformly and adequately enzymatically digested. After digestion, the brain tissues were transferred to Petri dishes containing trypsin inhibitor and DM and left to stand for 15 min. Again, the Petri dishes were shaken every 5 min to prevent further enzymatic digestion.

Note: Enzyme processing is also one of the most important aspects in cell culture. Not only is it critical to choose the right type of digestive enzymes, but also to get the time and temperature right. If enzymes are left at room temperature for a long period of time, their activity decreases over time. This is compensated for by one of the following methods in subsequent use: (1) increasing the enzyme action time; and (2) increasing the enzyme concentration. Therefore, the bottle containing the digestive enzymes should be placed in the refrigerator immediately after each use. Different enzymes should be selected according to the parameters of the tissue to be cultured. For example, the central annular ganglion, with its extensive connective tissue sheath, requires the selection of digestive enzymes that are more potent (e.g., proteases) and usually require a longer duration of action. Collagenase-hydrolase is generally considered to be the most effective because it contains two enzymes: collagenase digests collagenous connections between tissues optimally; and protease digests the sheath surrounding the ganglion. However, the choice of any given enzyme should be determined by 'trial and error'.

4. Hypertonic DM is prepared by pouring 750 fzl of an lmol/L glucose solution into 20 ml of DM in a large sterile beaker. this solution raises the osmolality of the DM from 130-145 mOsm to 180-195 mOsm. this hypertonic DM shrinks the neurons, thus making them "tougher" so that they can withstand the withdrawal process.

5. Brain tissue is transferred into dissecting trays containing the hypertonic DM and the central circumflex ganglion is secured.

6. Except during cell isolation and surgery, always cover the dissecting tray to prevent solvent evaporation. This is very important because solvent evaporation can lead to an increase in the osmotic pressure of the DM, causing cellular damage.

7. Attach appropriately sized Sigmacote-treated glass pipettes to the pipettes and sterilize them with ethanol for at least 5 min. rinse the microsyringes, tubes, and pipettes (IOml) thoroughly with ABS before filling them with hypertonic DM. It is also critical that there are no air bubbles in the tubes or microsyringes, as this will make cell extraction difficult.

8. Add the appropriate amount of medium (DM or CM 2.5-3 ml) to the cell culture dish and place the dish in the circular sorting compartment of a homemade plastic dish holder plate (Figure 12-2A). The cell culture dish used can be either a simple plastic product (3001) or a glass coverslip coated with poly-L-lysine affixed to the bottom of the dish. The latter is more suitable for observing the growth of neural protrusions because of the better optical clarity of glass compared to plastic. Although attaching the glass coverslip to the bottom of the plastic petri dish is laborious, this method does have several advantages. For example, any petri dish treated with antibiotics can be repeatedly reattached to a glass coverslip. Simply, a hole is drilled in the bottom of a 3001 Petri dish and the glass coverslip is attached with a non-toxic material that is rinsed with ultrapure water and treated with UV. It is important to apply unpolished, uncoated glass coverslips made from German glass (Bellco Glass, Inc. Biological Glassware and Equipment, USA). This petri dish provides the best optical resolution for both phase contrast microscopy and Nomarski optical microscopy.

9. Use fine forceps to remove the outer connective tissue sheath that surrounds each ganglion. To avoid hand shaking, try to place the forearm, or even the wrist, on the operating table and secure the fingers to the sides of the dissecting disk. Note that during the demyelination process, it is important to concentrate and maneuver meticulously.

10. (With fine forceps) Remove the inner connective tissue sheath that surrounds each ganglion. To prevent neuronal damage, the portion of the ganglion away from the desired neuron is usually pinched and gently torn. Care is taken to avoid touching the cytosol with the forceps to avoid cytosolic damage.

11. Using two forceps, gently squeeze the connection between the ends of the desired ganglion. This will sever the axons of most neurons and facilitate cell removal.

12. Move the tip of the pipette (using a micromanipulator) over the cell body and apply slight negative pressure through the microsyringe. The cell body will be gently sucked into the pipette. At this point, it looks like a balloon tied to a thin string, i.e., the cell body is the balloon and the axon is the string. Continue to apply slight suction (Fig. 12-2A), until the axon is abruptly pulled off and the cytosol enters the pipette.

13. When the cell has drifted into the pipette and is a few millimeters inside the opening, move the microscope (with the removable lever) and the light source so that the Petri dish is in the center (Figure 12-2A). Place the tip of the pipette against the bottom of the Petri dish and gently blow out the cells. If done accurately, undamaged cells will slowly settle to the bottom of the Petri dish.

Note: All transfers made between the dissection tray and the Petri dish should be accomplished by moving the cell traction device back and forth, taking care never to move the cell Petri dish.

14. Repeat steps 10 through 13 to obtain a sufficient number of cells. It is important to avoid physical interference with the cells in the dish during this process. In addition to chemical synaptic connections between neurons, neurons should always be spaced apart (5-10 cell diameters apart). If it is necessary to obtain solid chemical synapses, the cells should be discharged closely together.

15. Leave the dish (overnight) to allow the cells to attach, grow, and/or form synapses.

16. Remove instruments such as forceps, scissors, microsyringes, and test tubes from the bench, rinse with 70% ethanol, and store.

Results

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