Suny

Buffalo, New York 14214

"^Department of Physical Therapy D'Youville College Buffalo, New York 14201

^Neuroscience Center

University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27599

I. Introduction II. Maintaining Animals

III. Initial Dissection

A. Reagents and Supplies

B. Protocol

C. Comments

IV. Culturing Nerve Cells

A. Reagents and Supplies

B. Protocols

C. Types of Cultures

D. Comments References

The large neurons of the freshwater snail Helisoma trivolvis provide a unique preparation to study cytoskeletal mechanisms involved in neuronal growth and axon guidance. When placed into culture, these neurons form large growth cones in which cytoskeletal components and their dynamics can be analyzed with high-spatial resolution. Moreover, these growth cones display all of the dynamic features characteristic of growing axons, including advance, pause, collapse, and turning, allowing the correlation of cell biological mechanisms with growth cone

METHODS IN CELL BIOLOGY, VOL. 71 Copyright 2003, Elsevier Science (USA). All rights reserved. 0091-679X/03 $35.00

motility. This chapter describes complete procedures for culturing Helisoma neurons, including snail dissection, enzymatic treatments, removal of neurons, and necessary solutions, equipment, and supplies. Techniques are presented to culture Helisoma neurons by the extraction and transfer of individual neurons to culture dishes. A newer technique to dissociate neurons from whole ganglia is also described. In addition, methods to culture neurons on two substrates are presented. Culturing on polylysine in defined medium produces large, but nonmotile growth cones for cytoskeletal analysis, whereas culturing on polylysine in conditioned medium allows growth and motility for behavioral analysis. Recent tests suggest a new, simpler formulation for the medium used to culture Helisoma neurons that does not require the special-order medium that was previously used for cultures. These procedures make it feasible for someone inexperienced to successfully culture Helisoma neurons for use in a variety of experiments.

I. Introduction

Neurons of the freshwater pond snail Helisoma trivolvis exhibit the unusually large size characteristic of the molluscan phylum. The large diameter of neuronal cell bodies (up to 100 ym) and axons makes them uniquely accessible for the microscopic study of the neuronal structure and for the injection of probes to test neuronal function. In addition, the smaller number of neurons and their constant position in ganglia have allowed identification of many of the morphological, physiological, and molecular properties of individual neurons. This provides an opportunity to test repeatedly the responses of individual neurons with known identity and eliminates the variability inherent in populations of phenotypically mixed neurons. These neurons are also less dependent on growth factors for their short-term survival, which can be eliminated for some experiments. These properties make these neurons uniquely suited to a wide variety of cell biological questions, including growth cone formation during axon regeneration (Welnhofer et al., 1997; Ziv and Spira, 1997, 1998), mechanisms of growth cone motility during axon guidance (Zhou et al., 2002), synaptogenesis (Haydon, 1988), and cytoskeletal structure and dynamics (Cohan et al., 2001; Welnhofer et al., 1999; Zhou and Cohan, 2001).

Identified neurons from Helisoma buccal ganglia can be extracted with their original axons still attached to their cell bodies (Kater, 1974). When these neurons are plated onto polylysine-coated coverslips and cultured in defined medium (no growth factors), large growth cones (diameter about 50 ym) form within minutes at the tips of the axons. In addition, because of the high adhesion of the cell membrane to the substrate, these growth cones are not motile and do not collapse even when the actin filaments, which support the growth cone structure, are completely depleted (Zhou and Cohan, 2001). These properties allow a high-resolution study of cytoskeletal changes associated with growth cone responses to extracellular cues. In contrast, when the same identified neurons are cultured in medium conditioned with Helisoma brain, which provides growth factors and extracellular matrix, neurites emerge from the neuron cell body and the tip of the original axon. Small but motile growth cones are present at the tips of these newly formed neurites. The growth cones in conditioned medium share similar morphology and motile properties to vertebrate growth cones, which extend, turn, or collapse depending on the microenvironment they encounter. Previous studies have shown that growth cones formed on polylysine substrate or in conditioned medium respond to applied molecules with similar cytoskeletal changes (Williams and Cohan, 1994; Zhou et al., 2001, 2002). Therefore, by studying cytoskeletal rearrangement in the larger, nonmotile growth cones, it is possible to reveal the cytoskeletal mechanisms underlying the responses of the motile growth cones to environmental cues, such as collapse and turning (Zhou and Cohan, 2001; Zhou et al., 2002). Furthermore, after identifying the cytoskeletal changes in large nonmotile growth cones, the same changes can be verified in motile growth cones, bringing both findings together in one condition. Thus, by comparing the cytoskeletal reorganization of large nonmotile growth cones with cellular responses of motile growth cones, it is now possible to gain insight into cytoskeletal mechanisms underlying complex growth cone responses to extracellular factors with high resolution.

II. Maintaining Animals

Helisoma are maintained easily in 5-gal. aquaria, which provide good access to food and are easily handled and cleaned. All supplies can be obtained at local aquarium stores. Plastic aquaria with accompanying tops are convenient and have the advantages of resistance to breakage and they also provide a preferred surface (plastic) for egg laying, but glass aquaria are also used. Tanks should contain a layer of oyster shell or crushed coral about 1 cm thick on the bottom, which provides a source of calcium for shell development. Fill tanks with high-quality deionized water to which artificial salts (Instant Ocean) are added (1 g/gal.). The tanks should be aerated continuously, and a simple filter (corner or bubble type) should be used to remove debris from the water. Motorized filters should not be used because the vigorous water intake will remove newly hatched snails from the tank. The animals should be maintained on a 12-h light-dark cycle using a light timer and fluorescent lighting. Animals are fed Romaine lettuce and carrot slices as necessary. Water should be changed and the gravel rinsed at 2- to 3-month intervals, which encourages egg laying. Eggs are laid as small, circular patches on smooth surfaces. Embryos undergo a larval stage in the eggs and then hatch as small snails in about 8 days (Goldberg et al., 1988).

- III. Initial Dissection

In preparation for cultures, snails are anesthetized, their body wall is opened, and ganglia containing nerve cells are removed. Then ganglia are trypsin treated before the final dissection to remove nerve cells.

A. Reagents and Supplies

Helisoma saline (Table I) is used for animal dissections

0.15% trypsin (source: bovine pancreas; Sigma Chemical) diluted in medium in a 35-mm petri dish

0.2% trypsin inhibitor (source: soybean type I-S; Sigma Chemical) diluted in medium in a 35-mm petri dish.

Medium—Leibovitz L-15 is prepared so that the salt concentrations are appropriate for Helisoma (Table II). This has required a special order (Gibco; see later) in which L-15 powder is obtained without inorganic salts, which are then added at appropriate levels. Antibiotics (Gentamicin) may be added for long-term cultures. Because the L-15 special order is inconvenient (there is a 200-liter minimum order

Table I

Helisoma Saline"

Table I

Helisoma Saline"

NaCl

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