Pc12

Mitotic cell line

1000/cm2

14 days

B35 neuroblastoma

Mitotic cell line

4,500/cm2

5 days

"For some of these systems, the minimum achieveable cell density in culture has not yet been determined; these values are the minimum reported thus far. bSome of these systems have not been optimized for long survival in culture. The times listed may be exceeded with future modifications.

rat hippocampal neurons (Fletcher and Banker, 1989; Chapter 7), rat or mouse cortical neurons (Hayashi et al., 2002; Chapter 7), rat sympathetic neurons (Bruckenstein and Higgins, 1988; Lein et al., 1995; Wang et al., 1996), and chick forebrain neurons (Chapter 4). Essentially all neurons grown in culture also establish significant polarity by producing axons or at least the more generic processes referred to as neurites.

B. Intracellular Events

1. Cytoskeletal Transport and Dynamics

The movement, assembly, and disassembly of the cytoskeleton had been studied successfully in cell bodies, axons, and growth cones of a number of common o\

Table IV

Experimental Properties of Cultured Neurons

Name

Optical properties

Growth conditions

Chick sensory Good

Chick sympathetic Good

Rat sympathetic (SCG)

Chick ciliary Chick forebrain Rat hippocampal

Thin axons Good

Good Good Good

37 C, air incubator

37 C, air incubator

37 C, air incubator short term; COz incubator long term

37° C, C02 incubator

37 C, air incubator

37° C, C02 incubator

Rat cortical

Good

37 C, C02 incubator

Xenopus spinal neurons

Good

Room temperature (20-25° C)

Chick purkinje

Fair to good"

37° C, C02 incubator

Preparation requirements Difficulty of preparation

Overall cost

Timed fertile eggs Stereoscope Timed fertile eggs Stereoscope Timed pregnant rat Stereoscope

Timed fertile eggs Stereoscope Timed fertile eggs Stereoscope Timed pregnant rat Stereoscope

Timed pregnant rat Stereoscope

Gravid female, adult male frog Stereoscope Timed fertile eggs Stereoscope

Easy dissection

Easy dissection

Moderate dissection, handling neonates or adult female euthanasia Moderate dissection

Easy dissection

Moderate dissection Adult female euthanasia

Easy dissection Adult female euthanasia

Moderate dissection

Easy dissection

Low Low

Moderate

Low Low

Moderate if cell stocks are frozen Expensive if only fresh cells are used Low to moderate if cell stocks are frozen Moderate to expensive if only fresh cells are used Moderate to high

Heliosoma

Grasshopper sensory

Excellent for very large growth cones in non motile stateb Good for small growth cones in motile stateb Fair—in vivo preparation

Room temperature (20-25°C)

Helisoma brain extract needed for motile growth cones Room temperature (20-25°C)

Adult snail

Stereoscope

Moderate dissection

Grasshopper embryos Stereoscope

Moderate dissection

Low for small number of neurons

Low for single neuron

PC12 Good

B35 neuroblastoma Good

37 C, CO2 incubator 37° C, CO2 incubator

Tissue culture hood Tissue culture hood

None—cell line None—cell line

Low Low

"Astrocytes required in coculture for Purkinje cell survival and differentiation. Granule cell addition required for obtaining most mature phenotype. Neurons aggregate and processes fasiculate when cocultured with ED8 astrocytes, but single cells predominate when cocultured with ED16 astrocytes. bLarge growth cone on primary explanted axon is nonmotile and remains attached to poly-lysine-coated substrate even during collapse of the cytoskeleton. Small motile growth cones develop in the presence of Helisoma brain extract.

vertebrate neurons in culture. For example, studies involving light and electron microscopy have employed rat sympathetic superior cervical ganglion (SCG) (Wang et al., 2000; Roy et al., 2000; Yu et al., 2000), chick sensory (Bamburg et al., 1986), and Xenopus motor neurons (Chang et al., 1999; Chapter 8).

2. Exocytosis, Endocytosis, and Recycling

Endocytosis and endocytic recycling have been studied in the distal axon and growth cones of several cultured neurons, including chick dorsal root ganglia (DRG) (Overly and Hollenbeck, 1996), sympathetic (Bernstein et al., 1998), and ciliary ganglion (Diefenbach et al., 1999) neurons, the frog neuromuscular junction (Betz et al., 1992), and sensory neurons of Aplysia (Bailey et al., 1992).

3. Organelle Transport

The squid giant axon, while not strictly speaking an intact cell in culture, has been used extensively to study the fast axonal transport of organelles (Allen et al., 1982). It offers not only a good subject for light microscopy, but its size allows a variety of in vitro manipulations and provides adequate axoplasm to reactivate and observe organelle transport in vitro (Brady et al., 1985, 1993) and to carry out biochemical studies (e.g., Vale et al., 1985a,b). Organelle traffic has also been studied in the large bag cell neurons of Aplysia (Forscher et al., 1987; Savage et al., 1987) and in many smaller vertebrate neurons, including chick sensory and sympathetic (Hollenbeck, 1993; Morris and Hollenbeck, 1993, 1995) and rat sympathetic (Dailey and Bridgman, 1993) neurons. The study of organelle traffic in dendrites has of course been limited to cells such as hippocampal neurons (Overly et al., 1996) that produce dendrites in culture.

III. Choosing a Neuronal Culture System

Studies of the roles of specific proteins in cell differentiation and/or development often benefit from the generation of cell lines with stable expression of transgenes. While this is not possible for postmitotic neurons, several immortalized cell lines can be carried in culture indefinitely but can also be differentiated into neuron-like cells. Two of these, PC12 cells and B35 cells, are discussed in Chapters 12 and 13. While these cells have proven very useful in answering important cell biological questions about neuronal differentiation and development, there are no cell lines that fully differentiate into functional neurons. This section and Tables I-IV summarize the variables that make specific neuronal culture systems practical for various kinds of experiments and which are the most important in choosing which neuronal culture system to work with.

A. Basic Biological Properties

1. Dimensions

The size of the cell body, axon, and growth cone are important to consider for most experimental applications of cultured neurons (see Table II). Whether a neuron has desirable optical qualities and whether it is a good subject for direct manipulations such as electrophysiological measurements will depend at least in part on its dimensions. Commonly used neurons have cell body diameters that vary from the small neurons of the rat hippocampus, chick forebrain, or Droso-phila embryo, which are around 10 ^m, up to large invertebrate cells such as Heliosoma buccal ganglion neurons with diameters of 100 ^m. Not surprisingly, the cell body diameter is directly related to the axon or neurite diameter, which also varies widely from small vertebrate neurons with axon diameters <1 ^m to invertebrates such as Heliosoma (5-10 ^m) and upward to squid giant axon (500-800 ^m). It is important to consider that the projected diameter of the axon can be affected strongly by culture conditions: a wider, flatter axon better suited to light microscopic observation has been obtained by altering substratum conditions for Xenopus motor neurons (Chang et al., 1999) and by treating Drosophila neuroblasts with cytochalasin B (Saito and Wu, 1991). Growth cones, which can vary 10-fold in size among the neurons considered in this volume, likewise show significant variation in their size, shape, and behavior when the same neurons are grown on different substrata. In general, substrata that give the best spread and most readily observed growth cones also give the slowest outgrowth rates; e.g., the best substratum for observing the very large growth cones of Heliosoma allows virtually no motility (Chapter 9).

2. Growth Rate

The feasibility of studies of growth cone navigation, protrusive activity, or the growth cone cytoskeleton can depend on the rate and pattern of growth cone advance. These vary greatly, with growth cones of vertebrate peripheral neurons tending to move faster and more continuously than those of vertebrate central neurons, some of which advance in pulsatile fashion. Xenopus motor neurons have a very high rate of outgrowth, in vitro as in vivo. For a given type of neuron, the growth cone behavior and rate of advance typically depend strongly upon the exact culture substratum used (see later). Appropriate combinations of cell type and culture conditions can elicit the rapid initiation of growth cones after plating out (Chapters 2 and 3).

3. Length and Number of Neurites

Some of the neurons that form axons and dendrites in culture, such as hippo-campal, cortical, and forebrain neurons, tend to form one or two long axons, whereas neurons from sympathetic and dorsal root ganglia form variable numbers. This can depend strongly on the culture substratum, which can regulate not only the number of primary processes, but also the number and pattern of branches (Bray et al., 1987). This is a significant consideration for studies in which the number, geometry, or complexity of processes is deemed important. In addition, for studies of the signaling mechanisms and cytoskeletal events involved in neurite branching (Gallo and Letourneau, 1998), it may be useful to take advantage of intrinsic and induced differences among neurons in the frequency and pattern of branching.

4. Species

Selecting the species that will be the source for cultured neurons or cell lines can determine the effectiveness or cross-reactivity of reagents. Viral vectors, DNA constructs, antibodies, protein-loading procedures, or antisense RNA may work across species lines, but the variety of sources for neurons provides some control over this. The species may also be important if animal-holding facilities are needed. For example, an egg incubator or freshwater aquarium may be more readily available than a rodent facility, and it is certainly less expensive. In addition, genetically tractable species such as Drosophila (Chapter 11) or neuronlike cell lines such as PC12 (Chapter 12) or B35 cells (Chapter 13) that are subject to stable transfection can provide superior opportunities for manipulation of gene expression.

B. Developmental Properties

1. Rate of Development

Questions concerning cytoplasmic reorganization, cell polarity, and the origin of growth cones can best be studied using neurons that form growth cones rapidly after plating, such as those from the chick ciliary ganglion (Chapter 3) or the rat SCG (Chapter 2). Neurons that differentiate more slowly or that grow neurites at a slow but sustained pace for many days might be more useful for studies in which newly introduced genes need time to be expressed in sufficient amounts to affect growth properties.

2. Production of Dendrites

For some applications, such as studies of the cytoskeleton or mRNA localization, it is desirable to choose a neuronal cell type that produces bona fide dendrites in culture. As described earlier, the best systems for this application are rat hippocampal neurons (Fletcher and Banker, 1989; Chapter 7), rat cortical neurons (Chapters 7 and 19), rat sympathetic neurons (Lein et al., 1995; Chapter 2), and chick forebrain neurons (Chapter 4). The latter are less well characterized than the others, but their relative simplicity, purity, and high cell yield recommend them nonetheless.

3. Production of Synapses

Studies of cell-cell interaction, stimulated secretion, and synaptic transmission can be pursued in culture using either homologous or heterologous synapse-forming cultures. Chick sympathetic neurons can form axonal-somatic synapses within 2 days in culture, whereas rat hippocampal neurons, once they have developed dendrites with spines (2 weeks in culture), will develop many functional synapses between spines and axonal terminals. Chick ciliary ganglion neurons in high-density cultures will form large numbers of synapses within less than 12 h (Chapter 3).

4. Purity of Neurons in Different Preparations

Some sources of neurons, such as sensory ganglia, contain a significant fraction of glial or fibroblastic cells, even in early embryos. In cultures maintained for more than a day or two, these mitotic cells can overrun the neurons unless an anti-proliferative agent such as cytosine 1-^-arabinofuranoside (AraC) is used. Some neurons are sensitive to these agents, making their long-term use impractical, but most cultures will tolerate low but effective levels of AraC. There are sources of neurons such as chick forebrain or spinal cord that have vanishingly few nonneuronal cells and thus can be maintained as nearly pure cultures of neurons. The purity of primary neuron preparations is often critically dependent on the stage of embryo from which they are obtained. In some systems, nonneuronal cells can be removed efficiently from suspensions of tissue prior to plating out by mechanical methods (Chapter 19) or centrifugation (Chapter 5).

C. Source, Culture, and Handling Properties

1. Quantity of Neurons Available

The yield of neurons from a dissection procedure varies widely with the source (Table III) and must be high if neuronal cultures are to be used in biochemical procedures, metabolic labeling, or immunoblotting. For some applications, such as microscopy or single-cell polymerase chain reaction (PCR), it may actually be advantageous to have small numbers of neurons growing at low densities. Large invertebrate neurons are obtained in very small numbers, but each contains enough mass for PCR reactions and cloning. Some vertebrate sources, such as chick forebrain, can provide 108-109 cells from a relatively brief dissection. Peripheral ganglia, such as rat SCG and rat or chick DRG, as well as central nervous system (CNS) sources, such as chick spinal cord or rat cortex, provide intermediate numbers of cells, whereas rat hippocampus or Xenopus neural tube provide fewer still (Table III). Most neurons will grow in culture at a wide range of densities, and applications in this volume range from those requiring maximum cell mass per dish to those that require physically isolated neurons. It is common for neurons to tolerate high densities in culture (>5 x 104 cells cm2), providing adequate material for biochemical, metabolic labeling, or immunological procedures, as long as the necessary number of cells is available. Chick forebrain (Chapter 4) and rat cortex (Chapters 7 and 19) are particularly good systems for obtaining large numbers of relatively pure neurons and growing them at very high densities. However, many neurons will not grow well at extremely low densities. Hippocampal neurons are an exception, growing and differentiating well at densities as low as 103 cells cm2. Differentiated rat sympathetic neurons can be maintained at even lower densities. Chick forebrain and ciliary neurons will also grow at densities below 104 cells cm2. For many other neurons, experiments requiring single cells that have not contacted others must be carried out in the first few hours after plating, before extensive axonal growth causes cells to interact.

2. Culture Substratum

Many neurons can be grown on a variety of different substrata, ranging from tissue culture plastic to glass coated with organic polyions to more complex preparations of extracellular matrix molecules such as collagen or Matrigel. More elaborately prepared substrata may elicit more complex growth patterns and morphology than simpler substrata. However, in some cases, more desirable behavior may be produced by very simple substrata. For many vertebrate neurons, a simple polycation-coated substratum produces desirable properties such as large growth cones or complete differentiation of axons and dendrites. Many extracellular molecules such as laminin or fibronectin exert dramatic growth effects on neurons via receptor-mediated signaling. Thus for neurons cultured on simple substrata but in the presence of fetal bovine serum, the substrata may become altered via fibronectin binding. Many neurons and associated nonneuronal cells secrete materials that adhere to and modify the substrate so that even simple ones may become very complex over time (e.g., Mizel and Bamburg, 1976). These "microexudates" include glycosaminoglycans and proteoglycans.

3. Temperature and Atmosphere

Many vertebrate neurons require carefully controlled temperature for growth in the incubator as well as during observation on a microscope stage. For instance, chick and rodent neurons must be grown and handled at or near 37° C, whereas Xenopus and some invertebrate neurons can be maintained near ambient laboratory temperatures (Chapters 8 and 9). Vertebrate neurons also differ in their tolerance for air-buffered vs carbonate-buffered medium; the latter requires a CO2 incubator and/or observation chamber. While most laboratory tissue culture incubators are excellent at maintaining temperature and appropriate CO2 levels to hold culture pH, very little attention is usually paid to oxygen levels, which run about 18% in a normal CO2 incubator. In vivo, most vertebrate neurons experience oxygen concentrations in the 5-8% range, meaning that in vitro culture conditions are significantly hyperoxic. Recognition of the oxidative stress on neurons in culture, especially those from the CNS, has led to the development of antioxidant media and supplements that improve the long-term survival and health of the cells. The B27 supplement used commonly in culturing hippocampal and cortical neurons consists of enzymes for reducing superoxide and peroxide levels in addition to stimulants of the cellular antioxidant systems, such as reduced glu-tathione. However, even these supplements cannot overcome the oxidative stress put on hippocampal neurons cultured under a minimal layer (<3 mm) of medium. Specialized chambers and oxygen monitors are available for maintaining more physiological levels of oxygen through nitrogen displacement (e.g., Pro-ox model 110 from BioSpherix). Such systems are useful for applications in which low volumes of medium are required, such as the testing of expensive peptides or proteins that have to be added to cultures.

D. Ease and Cost of Obtaining and Growing Neurons

For many research questions in cell biology, there is likely to be more than one suitable neuronal culture system. If this is the case, then it only makes sense to use the one that is the least expensive to purchase, least expensive to house, easiest or least destructive to obtain, and easiest to grow and handle. This is an area in which neuronal cell lines look very attractive (Chapters 12 and 13). The following areas are worth considering (Table IV).

1. Suppliers

Most researchers have access to a supplier of timed pregnant rodents or day 0 fertilized chicken eggs. Those at universities with agriculture schools may find the availability and low price of the latter almost irresistible. However, acquiring freshwater snails or Xenopus may be just as easy in some locations. Do not underestimate the utility of an organism that is available anytime that you are ready.

2. Ease of Dissection

There is a large range in the relative ease of obtaining neurons by dissection from different sources. Although laboratories equipped with "good hands'' will have no trouble learning to dissect out most of the tissues described in this volume, some of the dissections, such as rat hippocampus, are more involved than others. For such dissections, a factor to consider is the availability of an experienced worker to teach the procedure to laboratory members. Obtaining most peripheral ganglia is a skill that can be learned in one or two sittings, whereas the dissection of chick forebrains is so easy that workers with a modest amount of experience can perform it without the aid of a dissecting microscope.

3. Freezing and Recovering Cells

Neuronal cell lines are stored routinely under liquid N2 and recovered into culture when needed. Although it is much less common to freeze and recover primary neurons, it is possible for some cell types and may be practical if each dissection is difficult or costly and provides far more cells than can be used immediately. The freezing of rat E18 hippocampal and cortical neurons (Chapters 7 and 19) provides from a single dissection a useful supply of neurons for many experiments. For example, one timed pregnant female rat typically carries 10 or more pups. These 10 pups will yield 5-6 x 106 hippocampal neurons and >108 cortical neurons each in a volume of several milliliters. This material can be divided into aliquots and frozen to good effect: the hippocampal dissection provides enough cells for a dozen or more typical cell biological experiments and the cortical preparation for even more, even for biochemical or immunological studies. Frozen hippocampal neurons have been recovered and kept in culture for several weeks and have successfully formed dendritic spines and neuronal networks that are indistinguishable from those made with freshly cultured cells. For some workers, this ability to maximize the products of a single dissection is what makes the rat CNS their system of choice.

4. Facilities

Different neuronal systems place different demands on animal facilities. Rodents require an animal house, chicken eggs only an incubator, and Xenopus or Heliosoma require freshwater aquaria. As researchers whose funds are consumed by animal maintenance know well, the security and cost of these facilities differ by orders of magnitude.

5. Bureaucracy and Karma

All researchers who employ animals in experiments must ask themselves how much of their time and effort they want to expend on animal care, and on filling out animal forms. Cracking open a fertilized chicken egg, dissecting a frog embryo, and sacrificing a pregnant female mammal are rightly considered to be very different by your institution's laboratory animal program, which may influence the choice of a culture system. In addition, other things being nearly equal, one always chooses a source for neurons that involves the destruction of the minimum number of adult animals, particularly cognizant ones.

IV. Conclusions

A wide range of cell biological and neurobiological questions can be addressed using neurons and neuron-like cell lines grown in culture, but a successful program of experiments requires the selection of the most appropriate culture system. This volume provides detailed information on the maintenance and use of more than a dozen diverse neuronal systems, and it is hoped that this introduction and its tabular data will allow workers to identify the culture system that most closely matches their needs and resources.

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