Fire Polished Glass Pipette

L-Glutamine

0.15 mg/ml

a The components (in mM) are added to L-15 (special order), which is purchased without inorganic salts. pH is adjusted to 7.5 and then the medium is sterile filtered.

a The components (in mM) are added to L-15 (special order), which is purchased without inorganic salts. pH is adjusted to 7.5 and then the medium is sterile filtered.

Fig. 1 Position of snail at start of dissection. The foot of the snail is placed against the dissecting pad, and pins are placed in two anterior and one posterior position to stabilize it. The incision (dotted line) is started at the ridge of skin just posterior to the foot and is extended anteriorly to the front edge. The cut is placed off midline to protect structures close to the surface.

at a cost of $400-$500 or more), an easier formulation has been tested. Standard L-15 medium is diluted to one-third strength, which has the appropriate concentrations of Na and K ions for Helisoma. Other salts are supplemented to their correct concentrations by adding 3.7 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES, and 0.15mg/ml L-glutamine and pH is adjusted to 7.5. Initial tests indicate that this medium supports viable Helisoma neurons. Growth cones that form in this medium in polylysine-attached cultures (see Section IV,C,1) are indistinguishable from those in special-order L-15.

Cell extraction medium—add 2 yl/ml of 1M CaCl2 to Helisoma medium Small scissors (12 cm length) for shell cutting

Vannas spring scissors—an inexpensive student grade such as #15100-09 from Fine Science Tools is adequate

Minutien pins 0.1 and 0.2 mm in diameter are available from Fine Science Tools (#26002-10 and #26002-20, respectively)

Dissecting dishes—Sylgard (Dow Corning) elastomer is an excellent dissection surface for pinning specimens. It can be purchased with black pigment, which provides the contrast necessary for visualizing specimens during dissection. A larger dish (60 or 100 mm petri) can be made for animal dissections and a smaller dish (35mm petri) can be made for ganglia dissection.

B. Protocol

1. Snails about 1 cm shell diameter are deshelled by cutting through their shell following its spiral curvature for one revolution. Cut with the tips of the scissors to avoid cutting the soft tissue of the snail body adjacent to the shell. The shell is then separated into halves and the snail is removed by gentle prodding with the tips of scissors, which must sever the attachment between the small columellar muscle and the shell.

2. The snail is placed in a solution of 25% Listerine (Pfizer) in saline for 20 min. In addition to antibacterial agents in Listerine, the menthol component acts as an anesthetic.

3. The snail is placed in a large, sylgard dissecting dish with its foot oriented downward and pinned (Fig. 1).

4. An incision is started about midway back on the dorsal surface of the snail where the skin (mantle) forms a lip. The incision is extended anteriorly to the front edge of the snail, progressing off the midline so that ganglia near the surface are not damaged.

5. The penis retractor muscle is cut and the penis is pinned to the side to allow access to ganglia. The esophagus is located as it originates from the buccal mass and passes through the brain (circumesophageal ganglia). It is cut posterior to the brain as it enters the area of the digestive system and then it is gently pulled forward through the brain and pinned anteriorly. This exposes the buccal ganglia and salivary glands that are positioned on the dorsal surface of the buccal mass (Fig. 2).

6. The last step involves cutting the attachments to remove the buccal ganglia and brain. The nerves and muscles that attach the buccal ganglia to the snail are cut, leaving as long a length as possible to facilitate pinning the ganglia later. However, the esophageal trunks, two nerves that innervate the esophagus (Fig. 2), should not be cut. They should remain attached to the esophagus, but their branches that innervate the salivary glands should be severed. Then the esophagus

Fig. 2 Diagram showing the placement of buccal ganglia (BG) and salivary glands (SG) on the buccal mass (BM). Nerves and muscle exit the buccal ganglia at its anterior, lateral, and posterior edges to connect to the buccal mass and other areas. Two long nerves, the esophageal trunks (ET), send branches to innervate the esophagus (ES) and salivary glands. Branches innervating the esophagus should remain intact, but those innervating the salivary glands should be cut. Bar: 700 ^m. (See Color Insert.)

Fig. 2 Diagram showing the placement of buccal ganglia (BG) and salivary glands (SG) on the buccal mass (BM). Nerves and muscle exit the buccal ganglia at its anterior, lateral, and posterior edges to connect to the buccal mass and other areas. Two long nerves, the esophageal trunks (ET), send branches to innervate the esophagus (ES) and salivary glands. Branches innervating the esophagus should remain intact, but those innervating the salivary glands should be cut. Bar: 700 ^m. (See Color Insert.)

should be cut at its junction with the buccal mass and the buccal ganglia and the esophagus is removed from the snail. Finally, the brain is removed by severing all its connections with the snail.

7. Ganglia are placed in the trypsin solution at room temperature. Buccal ganglia are incubated for 30 min and brains are incubated for 60 min.

8. After the incubation period, transfer the ganglia to the trypsin inhibitor solution and incubate for 25 min.

9. At the end of the trypsin inhibitor period, transfer the ganglia to cell extraction medium. Ganglia can remain in this medium for several hours before subsequent dissection, if necessary.

C. Comments

The initial dissection, from beginning of anesthesia to placement in trypsin, requires about 30 min for a snail and about 1.5 to 2h until the end of enzyme treatments. The effect of trypsinization is to allow the detachment of axons and nerve processes from connections in the neuropil of the ganglion during the extraction. Shorter incubation times (less than 20 min) result in cell bodies separating from their emerging axons as cells are extracted from the ganglia, which usually results in cell death. Although extensive testing with the new medium formulation (one third standard L-15) has not been conducted yet, initial results are positive. Neurons in polylysine-attached cultures are similar to neurons cultured in the special-order formulation. However, conditioned medium using the new formulation has not been tested yet, but it will be examined in the near future.

IV. Culturing Nerve Cells

A. Reagents and Supplies

Polylysine-coated coverslips—Coverslips are treated with polylysine to facilitate cell attachment. Acid-washed coverslips are incubated in a solution of 0.5% poly-L-lysine (MW 15,000-30,000, Sigma Chemical) in 0.15 MTris, pH 8.4, in a petri dish with gentle shaking for 60 min. Coverslips are then rinsed well one at a time in deionized water and subsequently incubated in phosphate-buffered saline (PBS) in a petri dish with gentle shaking for 60 min. The PBS is replaced and the incubation is repeated. Finally, coverslips are rinsed in deionized water individually and tilted against the edge of a large petri dish in a culture hood to dry. They may be stored as a single layer on filter paper at —20° C for 6-7 weeks.

Glass-bottom dishes—High-resolution optical studies require that cells be viewed on a coverglass substrate. Polylysine-coated coverslips may be attached to a suitable culture chamber in a variety of ways. A 15-mm hole can be bored in the bottom of a 35-mm petri dish or a chamber may be fabricated with a suitable hole. The coverslip can be attached to the chamber using a thin layer of silicon high vacuum grease. For more durable attachment, a thin line of 100% silicon rubber adhesive (Dow Corning) can be used (only types without mold inhibitors should be used). Alternatively, petri dishes with coverslips already attached may be purchased from MatTek Corp.

Micrometer syringe and assembly—This is necessary for delivering controlled amounts of positive and negative pressure during cell extraction. It can be purchased from Gilmont Instruments (0.2 ml size). The syringe is mounted on a short ring stand that also holds a three-dimensional micromanipulator (MM-33 or similar, Fine Science Tools). The syringe is attached to a hypodermic needle and plastic tubing that is connected to a glass micropipette. A plexiglass electrode holder in the micromanipulator clamps the micropipette in place. The micropipette is made from 1.5-mm O.D. borosilicate glass in a micropipette puller. The settings are adjusted so that the micropipette has a fairly long shank (1 cm), which can be placed close to ganglia without visual distortion. The end of the micropipette should be scored with a diamond scribe and broken so that the tip measures about 80-100 ym in diameter. It should be fire polished in a microforge (similar to those used for patch clamping) to obtain a smooth edge. The syringe, tubing, and micropipette must be filled completely with cell extraction medium without any air bubbles so that pressures can be applied instantly in the absence of a compressible filling. This is best accomplished by back-filling the syringe and tubing, attaching the syringe plunger, and then connecting the micropipette to the tubing.

B. Protocols

1. Extracting Single Neurons from Ganglia a. Ganglia (dorsal side up) are pinned to the black sylgard pad of a 35-mm petri dish using 0.1-mm minutien pins (Fig. 3). The black background provides the necessary contrast for visualizing the nerve cell bodies situated at the ganglionic surface, which are yellow within the orange background of ganglia. The best illumination is provided by a fiber-optic light source, which can be positioned close to ganglia. Ganglia should be stabilized well with pins to minimize movement during the subsequent dissection. Extracting nerve cells is a straightforward procedure, but it requires concentration and patience.

b. For smaller buccal ganglia, a small hole is made in the connective tissue capsule around the ganglia by pinching and gently tearing the capsule near the center of a ganglion using fine forceps (Dumont #5, Biologie tip). This procedure is critical for obtaining viable cells. It is important not to exert too much force so that the cells bulge out from the hole, which often results in death of cell bodies. Due to the strength of the capsule, it usually is not possible to tear it from one side to the other in one motion. Rather, the initial hole is enlarged periodically to access more neurons.

c. As neurons are exposed by this hole, the tip of the micropipette is positioned near a cell body and gentle suction is applied using the micrometer screw (Fig. 4). If suction is applied too quickly, cell bodies will separate from their emerging

Fig. 3 Preparing the buccal ganglia and brain for the extraction of neurons. (A) Buccal ganglia are removed from the snail and stabilized with pins through nerves, small portions of muscle (BMus), and esophagus attached to the ganglia. Neurons appear as large yellow spheres on the surface of ganglia, which have a background orange color. (B) After cutting the short cerebral commissure (*), the brain is pinned flat against the sylgard pad. Bars: (A) 500 ^m and (B) 300 ^m. (See Color Insert.)

Fig. 3 Preparing the buccal ganglia and brain for the extraction of neurons. (A) Buccal ganglia are removed from the snail and stabilized with pins through nerves, small portions of muscle (BMus), and esophagus attached to the ganglia. Neurons appear as large yellow spheres on the surface of ganglia, which have a background orange color. (B) After cutting the short cerebral commissure (*), the brain is pinned flat against the sylgard pad. Bars: (A) 500 ^m and (B) 300 ^m. (See Color Insert.)

axons and the cells will die. Suction is applied periodically while the micropipette tip is moved backward as the cell body is drawn out of the ganglion, still attached by its axon. Eventually, the axon may break and the cell body will move into the micropipette (Fig. 4B). Otherwise, the axon can be severed by advancing the micropipette (with the cell body and length of axon in its tip) into the sylgard pad.

d. After the cell body and axon are brought into the micropipette the nerve cell is transferred to the culture dish by positioning the micropipette tip near the coverslip bottom and applying positive pressure gently to expel the neuron (Figs. 4C and 4D). Then, another attempt to extract a new cell is made. The most successful technique for transferring nerve cells from ganglia to a coverslip involves a stationary culture chamber placed adjacent to the dissecting dish and a moveable microscope head and micromanipulator stand. After a cell body is taken into the micropipette, the microscope and micropipette are repositioned over the culture chamber so that the neuron can be expelled under visual observation.

e. Extracting neurons from the brain is performed similarly. However, the brain, which consists of nine, closely arranged ganglia, is larger, has a greater number of nerve cells, and the average size of a cell body is larger. This provides a greater opportunity for cultures and it can facilitate training in these procedures. The commissure connecting the cerebral ganglia should be cut so that ganglia can be pinned in a stable position (Fig. 3B). These ganglia contain a thicker, outer layer of connective tissue that should be removed with forceps. However, after this is accomplished, the ganglionic capsule can be torn more completely and more easily than the buccal ganglion capsule without damaging underlying nerve cells.

Fig. 4 Diagram of the procedure to extract individual neurons from ganglia. (A) A pipette tip is positioned near a hole in the ganglionic capsule. (B) Suction applied to the pipette draws a cell body and axon into the pipette tip. (C) The pipette tip is repositioned over the coverslip chamber. (D) Positive pressure through the pipette exzpels the neuron from the pipette and allows it to settle onto the coverslip where it adheres to the surface. (See Color Insert.)

Fig. 4 Diagram of the procedure to extract individual neurons from ganglia. (A) A pipette tip is positioned near a hole in the ganglionic capsule. (B) Suction applied to the pipette draws a cell body and axon into the pipette tip. (C) The pipette tip is repositioned over the coverslip chamber. (D) Positive pressure through the pipette exzpels the neuron from the pipette and allows it to settle onto the coverslip where it adheres to the surface. (See Color Insert.)

2. Dissociation of Whole Ganglia

We have developed a technique to dissociate the contents of entire ganglia, in contrast to extracting individual neurons as detailed in Section IV,B. Although the time required for preparing dissociated cultures is similar to that for extracting single neurons, dissociated cultures require less technical expertise and can be performed with little practice. To maximize the number of neurons in the cultures, Helisoma brains were used.

a. Two brains are removed from snails, the cerebral commissure is cut, and the brains are pinned so they lie flat in a sylgard dissecting dish. The outer, thick connective tissue surrounding the ganglia is removed partially with forceps or by cutting. Do not pierce the thin capsule that confines the neurons in each ganglion. Removing the thick connective tissue will facilitate dissociation later.

b. Brains are incubated in 0.3% collagenase (type XI, Sigma) in medium for 90min at 37° C.

c. Brains are incubated in 0.15% trypsin for 30-60 min at room temperature.

d. Brains are incubated in 0.2% trypsin inhibitor for 30 min at room temperature.

e. Brains are transferred to 0.5 ml cell extraction medium (2 yl/ml of 1 M CaCl2) for 15 min.

f. Brains are transferred to a tube containing 1 ml of medium and they are dissociated mechanically by passing them through the opening of a Pasteur pipette (tip reduced in diameter by fire polishing) 15-20 times.

Fire Polished Glass Pipette

Fig. 5 Neurons cultured on different substrates. (A) In polylysine-attached cultures, cell bodies and axons adhere quickly to the polylysine coating. A large growth cone forms at the cut end of the axon within 1 h. (B) Conditioned medium cultures promote neurite outgrowth from cell bodies and axons (C) over a 6- to 12-h period. Bar: 50 ym.

Fig. 5 Neurons cultured on different substrates. (A) In polylysine-attached cultures, cell bodies and axons adhere quickly to the polylysine coating. A large growth cone forms at the cut end of the axon within 1 h. (B) Conditioned medium cultures promote neurite outgrowth from cell bodies and axons (C) over a 6- to 12-h period. Bar: 50 ym.

g. The suspended neurons are transferred into one or more culture dishes, depending on the desired cell density.

C. Types of Cultures

1. Polylysine-Attached Cultures

In the simplest cultures, neurons are plated onto a polylysine-coated coverslip (Williams and Cohan, 1994; Welnhofer et al, 1997; Zhou et al., 2001, 2002) in medium in the absence of other extracellular matrix molecules or growth factors. Neurons with their attached axons stick immediately when they contact the coverslip and develop growth cones at the severed, distal end of their axons within a 1- to 2-h period (Fig. 5A). These growth cones are large (60-80 ym in diameter), do not advance, and their membrane remains attached to the coverslip even when their cytoskeleton is disassembled (Zhou et al., 2001, 2002). These properties make it

Fig. 6 Cultures of dissociated neurons. When neurons are dissociated from whole ganglia and placed into conditioned medium cultures, they form growth cones and neurites similar to neurons that are extracted individually from ganglia.

possible to perform high-resolution analyses of the neuronal cytoskeleton that are difficult or impossible in other preparations (Welnhofer et al., 1999; Zhou et al., 2002).

2. Conditioned Medium Cultures

When grown in conditioned medium cultures, Helisoma neurons produce neur-ites (Figs. 5B, 5C, and 6) and display the full complement of motile growth cone behaviors, including advance, collapse (Zhou and Cohan 2001), turning (Zhou et al., 2002), and synapse formation (Haydon, 1988). Growth cones in these cultures usually are significantly smaller than in polylysine-attached cultures, but they provide the important property of motility. Previous studies (Miller and Hadley, 1991; Barker et al., 1982; Wong et al., 1981, 1984) indicate that extracellular matrix molecules provide the signal for growth, similar to vertebrate neurons, but little is known about the identity of these molecules. Therefore, a conditioned medium that promotes growth is made by incubating brain ganglia for 72 h in coverslip-bottom (polylysine-coated) 35-mm petri dishes that contain four brains in 2ml of Helisoma L-15. After the incubation period, the brains are removed and neurons are plated directly onto the coverslip in these dishes. To facilitate cell attachment, which occurs more slowly in these cultures, neurons should remain undisturbed for the 8- to 12-h period required for outgrowth. Because sterility is an important consideration in these cultures, gentamicin can be added at a concentration of 50 yg/ml (Wong et al., 1981) at the start of the incubation period.

D. Comments

After neurons are placed into the culture chamber, adhesion to the substrate must occur for further development. Adhesion occurs quickly in polylysine-attached cultures but slowly in conditioned medium cultures. Adhesion is aided by maintaining the culture chamber in a fixed position so that neurons are not disturbed after they are deposited on the coverslip. Transferring neurons individually to a fixed culture chamber requires that the dissecting microscope is situated on a boom stand, which has an arm that can swing from dissection dish to the adjacent culture chamber. If this arrangement is not available, it may be possible to move the culture chamber gently in the case of polylysine-attached cultures, but this may be a problem for conditioned medium cultures.

Single neurons are deposited onto the coverslip surface by expelling them from the micropipette tip close to the coverslip. In polylysine-attached cultures, the intended result is growth cone formation at the tip of the axon. However, the manner in which neurons are expelled from the micropipette affects the position of the neuron as it attaches to the substrate and thus the ability to form growth cones. Due to the greater mass of the cell body compared to the axon, cell bodies will sink toward the coverslip faster and adhere on contact, causing the axon to project up into the medium. In this situation, growth cones will not form. However, by expelling neurons closer to the coverslip, small streams of positive or negative pressure can be used to orient the axon and its tip to ensure contact and adhesion to the substrate. Precise placement of neurons is not necessary for conditioned medium cultures because growth cones and ensuing neurite outgrowth occur from the cell body as well as from axons in these cultures (Figs. 5B and 5C). Visualization of cell bodies and axons on the coverslip is enhanced by indirect illumination, which allows neurons to appear bright against a dark background.

Finally, it is also important to consider potential difficulties with these cultures. Cross-reactivity of Helisoma antigens to antibodies from vertebrate species can limit immunocytochemistry in these cultures. In addition, due to the smaller number of neurons in the snail nervous system, these cultures contain fewer nerve cells. Cultures prepared by extracting individual neurons typically contain less than 15 neurons. However, this smaller number of cells is justified given the depth of cytoskeletal analysis that can be attained. This may be a disadvantage for other types of experiments that require assays of large populations of neurons. Dissociated Helisoma cultures containing larger numbers of neurons may overcome some of these difficulties. Finally little information exists on genetic manipulations necessary for the expression of molecules in snail neurons, although this methodology should be feasible as shown for a wide variety of species. Despite these limitations, Helisoma cultures provide one of the few neuronal preparations where cytoskeletal mechanisms can be visualized and manipulated to gain insight into many cell biological questions.

Acknowledgments

The authors acknowledge the pioneering work of Dr. S. B. Kater who initially developed Helisoma as a model system to study neuronal function. This work was supported by funds from the NIH and NSF to CSC.

References

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