Neuron Replating

Ingredient

Amount Final concentration

N2 medium

100 ml 100% (v/v)

OP-1 (Curis)a

5 ^l (from 1 mg/ml stock) 50 ng/ml

aTo the best of our knowledge, OP-1

is only available by collaborating with Curis (www.curis.com).

IV. Maintenance of Sympathetic Neuronal Culture

A. The day after plating, replace N2 medium with medium A.

B. The next day, replace two-thirds of medium A with medium B. Repeat every other day.

C. One week after plating, start feeding the culture with medium C. Each time, replace two-thirds of the medium with medium C.

D. After 7-10 days in medium C, the cells will have well-developed dendrites.

IV. Maintenance of Sympathetic Neuronal Culture

A. The day after plating, replace N2 medium with medium A.

B. The next day, replace two-thirds of medium A with medium B. Repeat every other day.

C. One week after plating, start feeding the culture with medium C. Each time, replace two-thirds of the medium with medium C.

D. After 7-10 days in medium C, the cells will have well-developed dendrites.

V. Methylcellulose-Thickened Medium Inert thickening reagent methylcellulose can be added into either L-15 incomplete medium or N2 medium to facilitate cell attachment to relatively less adhesive substrates, e.g., plain glass. A variety of different types of methylcellulose are available commercially. We use F4M premium grade methocel from Dow Chemical Company (Midland, MI). The procedure for preparing 0.6% methylcellulose containing medium is as follows: Weigh out 0.3 g methylcellulose into a 50-ml polypropylene tube. Autoclave. Add 50 ml L-15 plating medium or N2 medium. Turn the tube upside down and bang the cap against a tabletop to loosen the methylcellulose at the bottom and let it float in the medium. Attach the tube to an electric rotator and rotate at 4 C for at least 12 h to completely dissolve methylcellulose. Warm up when ready to use.

nerve growth factor, which is required for the viability of the cultures, unless replaced with alternative growth factors such as brain-derived neurotrophic factor (Ernst et al., 2000).

Either type of medium can be supplemented with an inert thickening agent called methylcellulose, which has certain advantages but also has disadvantages depending on the specific needs of the experiment. One main advantage is that cells cultured in methylcellulose will adhere to plain glass coverslips or plastic dishes that are not treated with any substrate or positively charged molecule such as poly-D-lysine. The neurons are not adhered tightly under these conditions, but they are sufficiently adhered to support axonal outgrowth. Cultures of this kind are particularly useful for experiments involving axonal retraction, which is more robust when the axons are initially not adhered so tightly to the substrate (see, e.g., Shaw and Bray, 1977; Ahmad et al., 2000; He et al., 2002). Methylcellulose presumably promotes attachment to poorly adherent substrates simply by "weighing" the cells down and permitting them to remain stationary long enough to form attachments. Another advantage of methylcellulose is that it tends to dissuade neuronal cell bodies from clumping together. We have encountered two disadvantages of methylcellulose. First, it is more difficult to exchange media or to introduce drugs into the medium when methylcellulose is present; this must also be done using medium containing methylcellulose so that the two media are miscible. Second, we have found that the cells sometimes extract unevenly in the presence of methylcellulose; for example, in immunofluorescence preparations. Instructions for including methylcellulose in the medium are also provided in Table 3.

C. Dissection

1. Chick Sensory Ganglia

Fertilized chicken eggs are incubated at 37°C in a humidified egg incubator. Lumbosacral DRGs are dissected from the embryos after 10-11 days of incubation. Eggs are wiped with 70% ethanol and are cracked into a sterile petri dish. The chicken embryo is separated from other contents with sterile forceps and scissors. The head is removed, and the rest of the embryo is placed on a dissecting plate with the ventral side facing up. Syringe needles are used to fix the embryo against the dissecting plate. Legs are stretched laterally to flatten the embryo. The next steps are performed under a dissecting microscope. Two pairs of forceps are used to gently open trunk skin and muscle, and the chest wall. Organs in the chest and abdomen are gently removed with forceps, with care not to damage the spinal column area. The tail is cut off to reduce fluid accumulation in the abdomen. We then increase magnification at the microscope for a better view of the area containing the lumbosacral DRGs. The connective tissue is removed carefully to expose clearly the spinal column, the lumbosacral DRGs, and spinal nerves on both sides (see Fig. 1). The chain of sympathetic ganglia sits along the lateral side of the spinal column and on top of (i.e., ventral to) the DRGs. The chain is removed carefully, with care not to damage the DRGs. The lumbosacral DRGs are dissected using two pairs of fine-tip forceps, with one lifting the spinal nerve bundles and the other disconnecting the DRGs from the spinal cord. The DRGs are then transferred immediately to a petri dish containing Leibovitz's L-15 medium. (For this, we use L-15 directly from the bottle, with no supplements.) The DRGs are then separated from spinal nerves and cleared of any covering tissues. Generally speaking, three to five DRGs can usually be obtained from each side of the embryo.

2. Neonatal Rat Superior Cervical Sympathetic Ganglia

Pregnant female Sprague-Dawley rats generally give birth on the 21st or 22nd day postconception. Litters usually consist of 8-14 pups. We dissect superior cervical sympathetic ganglia from postnatal day 0 or day 1 rat pups. Pups are buried under watery ice for at least 15 min to achieve anesthesia. Pups are then sprayed with 70% ethanol and fixed to a wax dissecting plate with three syringe needles (Fig. 2A). A pair of small scissors is then used to cut the heart (entering through the underarm region). This drains blood from the pup, which is critical for achieving a clear dissecting view for the later procedures. The skin is then cut open along the midline from the midthorax to mandible level and from the lower cervical level to the two upper limbs. Under a dissecting microscope, two large salivary glands are clearly seen along the midline of the neck (Fig. 2B). One of the salivary glands (with surrounding connective tissue) is removed with a pair

Fig. 1 Dissection of DRGs from a day 10 chicken embryo. After decapitation, the embryo was pinned to the dissection plate with its ventral side up. Organs in the chest and abdomen were removed. Working under a dissecting microscope, connective tissues surounding the spinal column and DRGs were removed carefully. Shown in the middle of the view in this figure is the lumbosacral region of the spinal column (the top of the photo is rostral). On the left side of the figure, lumbosacral DRGs and their roots are highlighted in pink and the sympathetic chain is highlighted in blue. The sympathetic chain has been removed on the right side of the figure to expose the DRGs beneath. Arrows point to three DRG. (See Color Insert.)

Fig. 1 Dissection of DRGs from a day 10 chicken embryo. After decapitation, the embryo was pinned to the dissection plate with its ventral side up. Organs in the chest and abdomen were removed. Working under a dissecting microscope, connective tissues surounding the spinal column and DRGs were removed carefully. Shown in the middle of the view in this figure is the lumbosacral region of the spinal column (the top of the photo is rostral). On the left side of the figure, lumbosacral DRGs and their roots are highlighted in pink and the sympathetic chain is highlighted in blue. The sympathetic chain has been removed on the right side of the figure to expose the DRGs beneath. Arrows point to three DRG. (See Color Insert.)

of forceps. This exposes the ipsilateral sternocleidomastoid muscle (Fig. 2C). Transection of this muscle exposes the strap muscles covering the vessels beneath. Dissecting these muscles will then expose the carotid artery. Under higher magnification, bifurcation of the carotid artery can be resolved; the nodose ganglion is located directly lateral to the bifurcation (Fig. 2D). The nodose ganglion is removed to avoid confusion with the sympathetic ganglion later. The superior cervical ganglion sits directly beneath (i.e., dorsal to) the carotid bifurcation. There are two ways to dissect out the sympathetic ganglion. In the first way, the dissector lifts the tail of the ganglion and the carotid artery together with one pair of forceps and then separates the ganglion and the artery from surrounding tissues with another pair of forceps. Once fully detached, the ganglion and the artery are transferred into Leibovitz's L-15 medium in a petri dish. In the second way, the carotid artery and its branches are removed first, which will clearly expose the sympathetic ganglion beneath. Then two pairs of forceps are used to dissect out

Fig. 2 Dissection of superior cervical ganglion from neonatal rat. (A) A neonatal rat pup was fixed on a dissecting plate with three syringe needles after anesthesia. (B) The two large salivary glands (one of which is outlined by arrowheads) along the midline were exposed after the neck skin was cut open. (C) After the salivary glands were removed, the sternocleidomastoid muscle (bracketed by arrows) was revealed on both sides. (D) Transection of the sternocleidomastoid muscle and strap muscles beneath has exposed the carotid artery and its branch (shown at higher magnification). The nodose ganglion (indicated by the arrowhead) is located directly lateral to the branch point of the carotid artery, and the sympathetic ganglion (indicated by the arrow) is located directly beneath the branch point.

Fig. 2 Dissection of superior cervical ganglion from neonatal rat. (A) A neonatal rat pup was fixed on a dissecting plate with three syringe needles after anesthesia. (B) The two large salivary glands (one of which is outlined by arrowheads) along the midline were exposed after the neck skin was cut open. (C) After the salivary glands were removed, the sternocleidomastoid muscle (bracketed by arrows) was revealed on both sides. (D) Transection of the sternocleidomastoid muscle and strap muscles beneath has exposed the carotid artery and its branch (shown at higher magnification). The nodose ganglion (indicated by the arrowhead) is located directly lateral to the branch point of the carotid artery, and the sympathetic ganglion (indicated by the arrow) is located directly beneath the branch point.

the ganglion and transfer it to Leibovitz's L-15 medium in a petri dish. The ganglion is cleaned of blood vessels and covering connective tissues with two pairs of fine-tip forceps. The nerves from the ganglion are severed from the body of the ganglion. Then, the entire procedure is repeated on the other side of the animal to obtain the second superior cervical ganglion.

D. Preparation of Dissociated Cultures

After dissection from the animals and cleaning of any contaminating tissue, chick DRGs and rat superior cervical ganglia are treated in a very similar way to obtain cultures of dissociated neurons. The sympathetic ganglia are transferred to a small tube containing 3 ml of 2.5 mg/ml collagenase (Worthington Biochemical), in calcium and magnesium-free phosphate-buffered saline (PBS) and incubated at 37°C for 15 min. The ganglia are then incubated in 2.5 mg/ml trypsin (Worthington Biochemical) in PBS at 37°C for 45 min. (If sympathetic ganglia are dissected from rat fetuses, the collagenase incubation may not be necessary because younger ganglia are easier to dissociate than older ones.) It is important to make sure that all of the ganglia sink to the bottom of the tube, as sometimes they can stick to the edge of the tube or float on a small air bubble. For chick DRGs, we incubate the ganglia with both enzymes at the same time for 20 min at 37°C. At the end of the enzyme treatments, the DRGs or sympathetic ganglia are rinsed twice in L-15 blocking medium (for composition, see Box 3) to neutralize the enzymes. Each rinse is for 5 min. For cultures that will be eventually grown in serum-free N2 medium, the ganglia are rinsed two more times in N2 medium (see Box 3) to remove any residual fetal bovine serum (FBS). We then add about 1 ml of complete L-15 plating medium (see Box 3) or N2 medium. The ganglia are then triturated into a single-cell suspension with a Pasteur pipette whose tip has been fire polished to obtain a narrow opening. The diameter of the pipette opening should be slightly smaller than that of the ganglia so that the ganglia are squeezed through it and dissociated sufficiently. In our experience, trituration should be done roughly five or six times. The concentration of cells in the trituration medium can be counted on a hemocytometer and diluted in L-15 plating medium or N2 medium to the desired concentration for plating. Alternatively, one can determine empirically the number of ganglia required to generate a desired cell density in a certain number of cultures. Cells cultured in L-15 plating medium are kept at 37°C in normal air, whereas cells cultured in N2 medium are kept at 37° C in 5% CO2. When dividing the cells into dishes, it is important to work quickly because dispersed cells can settle quickly in the test tube or the pipette, resulting in cultures of very different density from one another.

E. Maintenance of Cultures

Neurons cultured for more than 2 days must be "fed" roughly every other day or twice a week. For this, we remove two-thirds of the medium and replace this volume with fresh medium. The inclusion of serum in the medium will promote dendritic differentiation in cultures of sympathetic neurons, but even more rapid and robust dendritic differentiation can be induced by including a member of the transforming growth factor fl family called OP-1 (see, e.g., Yu et al., 2000). Cytosine arabaninoside (also called Ara-C or 1-fl-D-arabinofuranosylcytosine), a deoxycytidine analogue, can be added into the culture to inhibit the growth of dividing nonneuronal cells, which could dominate the culture otherwise. B-27, a commercially available mixture of culture additives, contains a cocktail of anti-oxidants that reduce reactive oxygen damage and enhance optimal growth and long-term survival of neurons. Details on our standard method for maintaining

Dendrite And Axon Culture
Fig. 3 Sympathetic explant culture. A chunk of neonatal rat sympathetic ganglion was plated on a substrate of poly-D-lysine and laminin. One day after plating, numerous axons had grown radially from the cell body mass (CBM). Arrows point to a few nonneuronal cells.

long-term sympathetic cultures and inducing dendrite formation are provided in Table 3.

F. Preparation of Explant Cultures

Explants are another way to culture neurons. Rather than dissociating the ganglia into individual cells, the ganglia are simply cut into small pieces and cultured as chunks. Axons grow radially away from the "cell body mass.'' Cultures of this kind are used far less commonly than dissociated cultures because the morphology of individual neurons cannot be fully discerned. However, there are special applications for which explant cultures are ideally suited. For example, explant cultures can be used for biochemical analyses in which one wishes to compare composition of the axons with that of cell bodies and dendrites. The cell body mass can simply be cut away from the axons under a dissecting microscope and then plucked out of the culture after development of the axonal halo. The axonal halos and cell body masses can then be analyzed separately by biochemical means. The axonal halo contains only axons, whereas the cell body masses contain cell bodies, proximal regions of the axons, and, in the case of sympathetic neurons, dendrites. For a clean separation, it is necessary that there are no loosened cell bodies within the axonal halo. If this happens, the cell body masses can be plucked out and then cultured a second time, which results in a cleaner axonal halo. Most typically, a collagen substrate is used for explants because the explants do not tend to adhere well unless they have a thick substrate into which to "burrow." However, smaller explants can be grown successfully on any of the substrates used for dissociated cultures. Fig. 3 shows a small explant grown on laminin for 1 day; axonal outgrowth is apparent, as are a few nonneuronal cells that have detached from the cell body mass. More details on explant cultures are provided in Peng et al. (1986).

G. Reducing Nonneuronal Contamination

For most purposes, it is advantageous to have cultures that are as enriched as possible for neurons. However, ganglia are composed of nonneuronal cells (fibro-blasts and glial cells), as well as neurons, and hence primary neuronal cultures consist of a mixture of cell types. Because nonneuronal cells undergo mitosis, whereas neurons do not, ganglia from younger animals contain a higher proportion of neurons to nonneuronal cells than ganglia from older animals. One method for routinely obtaining "cleaner" cultures of neurons is to carefully remove the capsule from each ganglion after it has been dissected from the animal. The capsule surrounding each ganglion is essentially invisible to the eye, but can be pealed away (almost like a banana peel) by a talented dissector with some good luck. An attempt should be made, but if the ganglia begin to tear, it is wise to abandon the attempt to remove the capsule and simply live with additional nonneuronal cells in the culture. Tearing a ganglion can result in the death of many neurons. Some workers have found that they can reduce nonneuronal contamination by plating the cells, agitating the culture, and replating the cells that lose their attachment during agitation (Shaw and Bray, 1977). Neurons are more poorly adhered than nonneuronal cells, and hence replating will result in a richer density of neurons. We have not had good luck with this approach, although we have not put substantial effort into it. For long-term cultures, antimitotic agents such as cytosine arabinoside can be added to the cultures. These agents prevent cells from dividing and slowly kill cells that normally divide, but do not have notable adverse effects on neurons if used at appropriate concentrations. For dissociated cultures, a concentration of 0.24 yg/ml generally works well; this concentration can be increased 10-fold for explant cultures (Peng et al., 1986). For experiments on cultures that are grown for less than 3 days, there is no point in adding such agents. However, for sympathetic cultures grown for over a week, exposure to cytosine arabinoside can result in cultures that consist of over 95% neurons. This is particularly useful for biochemical studies. Eliminating nonneuronal cells from DRG cultures tends to be more problematic, for reasons that we do not understand.

Chick Forebrain Neurons

Fig. 4 Dissociated embryonic chick DRG culture. Embryonic day 10 chick DRG neurons were plated on plain glass coverslips in L-15-based medium thickened with methylcellulose. By 16 to 20 h after being plated, DRG neurons had generated axons tipped with growth cones. Some neurons generate several simple axons without branches (A), whereas others generate axons that bifurcate (B).

Fig. 4 Dissociated embryonic chick DRG culture. Embryonic day 10 chick DRG neurons were plated on plain glass coverslips in L-15-based medium thickened with methylcellulose. By 16 to 20 h after being plated, DRG neurons had generated axons tipped with growth cones. Some neurons generate several simple axons without branches (A), whereas others generate axons that bifurcate (B).

Fig. 5 Rat sympathetic neuronal culture. (A) When plated in L-15-based medium on a poly-D-lysine-coated coverslip, neurons attach firmly to the substrate and generate small lamella and numerous filapodia. Most neurons do not grow processes under these conditions. (B) When plated in N2 serumfree medium, sympathetic neurons show somewhat more robust outgrowth, with broader lamellae and short stumpy processes with large growth cones.

Fig. 5 Rat sympathetic neuronal culture. (A) When plated in L-15-based medium on a poly-D-lysine-coated coverslip, neurons attach firmly to the substrate and generate small lamella and numerous filapodia. Most neurons do not grow processes under these conditions. (B) When plated in N2 serumfree medium, sympathetic neurons show somewhat more robust outgrowth, with broader lamellae and short stumpy processes with large growth cones.

III. Cultures

In closing, this section is devoted to describing the cultures that result from the efforts described earlier.

Bad Neuronal Culture

Fig. 6 Laminin-induced axogenesis from sympathetic neurons. Sympathetic neurons were treated with laminin in N2 serum-free medium. (A and B) Thirty minutes after laminin treatment, broad lamellae have extended from the base of the cell bodies. Early processes tipped with large growth cones have begun to protrude from the periphery of the lamellae (arrowhead) and some short axons are already seen (arrow). (C) By 3 h after laminin treatment, sympathetic neurons had generated long, thin axons with complex branches.

Fig. 6 Laminin-induced axogenesis from sympathetic neurons. Sympathetic neurons were treated with laminin in N2 serum-free medium. (A and B) Thirty minutes after laminin treatment, broad lamellae have extended from the base of the cell bodies. Early processes tipped with large growth cones have begun to protrude from the periphery of the lamellae (arrowhead) and some short axons are already seen (arrow). (C) By 3 h after laminin treatment, sympathetic neurons had generated long, thin axons with complex branches.

A. Short-Term Chick Dorsal Root Ganglia Culture

Chick DRG neurons are generally used for short-term experiments in our laboratory. We typically plate DRG neurons in methylcellulose-thickened L-15 plating medium on plain glass coverslips. Because plain glass is a relatively less adhesive substrate, these conditions are ideal for studying axonal retraction (Ahmad et at., 2000; He et at., 2002). By 16 to 20 h after plating, most DRG neurons have grown several distinguishable axons tipped with growth cones. The axonal identity of these processes is evidenced by their long, thin, and

Insect Primary Neurons Culture
Fig. 7 Long-term rat sympathetic culture. Neonatal rat sympathetic neurons were plated and maintained as described in the text. A typical neuron is shown. After roughly 2 weeks in culture, the neuron has developed several short, thick and tapering dendrites, one of which is marked by an arrow.

uniform-diametered morphology, and also by their expression of axonal marker proteins (Smith, 1998). Some DRG neurons generate several simple primary axons (Fig. 4A), whereas some generate primary axons that bifurcate (Fig. 4B). When cultured for a longer period of time, a typical DRG neuron undergoes significant recrafting of the axonal arbor to produce a morphology that better reflects that of the DRG neurons inside the animal.

B. Short-Term Rat Sympathetic Culture

For most of the short-term experiments in our laboratory, we plate sympathetic neurons on poly-D-lysine-coated glass coverslips in L-15 plating medium. Neurons attach to the bottom of the dish shortly after being plated. Within the first 1 to 2 h after plating the neurons have generated a modest lamella from the base of the cell body, with numerous filopodia extending from the edge of the lamella (Fig. 5A). By this time, neurons have attached to the coverslip firmly. The morphology of the neuron remains essentially the same over the next several hours and even by the next morning. The rapid outgrowth of axons can be induced by the addition of laminin to a final concentration of 25 yg/ml (Invitrogen) to the culture (Rivas et al.,

1992; Tang and Goldberg, 2000). A commercially available partially defined mixture of growth factors (which contains laminin as well as other factors) termed Matrigel matrix (BD Biosciences) can be used at 1:40 dilution instead of pure laminin for an even more rapid and robust response (Slaughter et al., 1997; Yu et al., 2002). Alternatively, laminin can be applied directly onto the poly-D-lysine substrate prior to the plating of the cells (see Box 2). This approach produces a rapid outgrowth of axons that begins almost immediately after the firm attachment of the cell body to the substrate (roughly 30 min after plating; see, e.g., Ahmad et al., 1999). When grown in the presence of laminin or matrigel, neurons generate thin and extremely long axons, as shown in Fig. 6. It is our impression that an even broader, more robust lamella is produced on the poly-D-lysine and that an even more robust response to laminin or matrigel can be obtained by using the N2-based medium as opposed to the L-15-based medium (see Fig. 5B). We generally use the L-15-based medium, however, because its capacity to maintain pH in normal air is advantageous for experimental manipulations on the microscope stage.

C. Long-Term Rat Sympathetic Culture

Neurons in culture generate dendrites much later than axons. Therefore we need to maintain rat sympathetic neurons in culture for a longer period of time to study dendrites. Generally speaking, after 7 to 10 days in medium containing OP-1, neurons will have developed robust dendrites (Yu et al., 2000). Each neuron has several dendrites, which are distinguishable from axons on the basis of their short, thick, and tapering morphology (Fig. 7). The axons have become extremely long at this point and have formed a dense network on the culture substrate. It is impossible to discern at this point which axons arise from which cell bodies, but interestingly, the number of axons has been pruned such that each individual neuron typically has a single axon. This can be revealed by microinjecting a fluorescent dye into an individual cell body to reveal its detailed morphology (Higgins et al., 1991).

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