The Culture of Chick Forebrain Neurons

Steven R. Heidemann, Matthew Reynolds, Kha Ngo, and Phillip Lamoureux

Department of Physiology Michigan State University East Lansing, Michigan 48824

I. Introduction

II. Growth and Development Characteristics

A. Chick Forebrain Neurons Develop into Typical Pyramidal Neurons in Low-Density Culture

B. Chick Forebrain Neurons Show Elongation Behaviors Similar to Hippocampal Neurons

C. Chick Forebrain Neurons Differ in Important Ways from Hippocampal Neurons

III. Isolation of Single Chick Forebrain Neurons

A. Obtaining and Maintaining Chick Embryos

B. Dissection of the Forebrain

C. Dissociation of Forebrain Tissue

IV. Culture Conditions for Chick Forebrain Neurons

A. Polylysine Treatment of Tissue Culture Surfaces

B. Culture Media

C. Plating Efficiency and Culture Density

D. Improvements for the Future References

Dissociation of the forebrain of a single 8-day chick embryo produces >107 neurons in nearly pure culture. Our methods allow 50-70% of these neurons to develop an axon and typical pyrimidal shape after 3-4 days in culture at low density (104 cells/cm2) by a stereotyped developmental sequence similar to that of rat hippocampal neurons. The culture method for chick forebrain neurons is unusually rapid, inexpensive, simple, and could be used in undergraduate laboratory exercises. The dissection and dissociation of the tissue are easy and

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

rapid, requiring less than 30 min from cracking open the chicken egg to plating the cells. Axonal development by these neurons and growth for about a week do not require glial support. The neurons are grown on polylysine-treated culture surfaces in either CO2-dependent (Medium 199) or -independent (Liebovitz L15) media with 10% fetal bovine serum and a supplement based on the classic N2 supplement for neuronal culture.

I. Introduction

The culture of forebrain neurons from embryonic chicks at day 8 (E8) is notable for the rapidity, ease, and economy of obtaining very large numbers of neurons with highly stereotyped development of a typical pyrimidal shape (Fig. 1).

Neurons Cell Culture

Fig. 1 Axonal morphogenesis by chick forebrain neurons in culture. (A) Phase micrograph of cells approximately 4 h after plating. Most cells have short filopodial and/or lamellipodial extensions surrounding the margin of the cell. We call this stage 1, as for rat embryonic hippocampal neurons. (B) After 1 day in culture, most cells have developed minor processes and thus developed to stage 2. A few neurons have begun axonal outgrowth, e.g., the neuron in the lower left, and so advanced to stage 3. (C) Lower magnification view of a culture after 3 days: 40-50% of neurons have developed an axon and developed to stage 3. (D) After 5 days the fraction of cells developing to stage 3 has leveled off at 50-70% of neurons. After day 5, all neurons begin to degrade when cultured in L15 medium (see Section IV,B). In Medium 199, stage 3 neurons remain healthy for another 5-7 days, but cells that do not develop to stage 3 undergo gradual attrition. Bar: 22 ^m (A and B) and 44 ^m (C and D).

Fig. 1 Axonal morphogenesis by chick forebrain neurons in culture. (A) Phase micrograph of cells approximately 4 h after plating. Most cells have short filopodial and/or lamellipodial extensions surrounding the margin of the cell. We call this stage 1, as for rat embryonic hippocampal neurons. (B) After 1 day in culture, most cells have developed minor processes and thus developed to stage 2. A few neurons have begun axonal outgrowth, e.g., the neuron in the lower left, and so advanced to stage 3. (C) Lower magnification view of a culture after 3 days: 40-50% of neurons have developed an axon and developed to stage 3. (D) After 5 days the fraction of cells developing to stage 3 has leveled off at 50-70% of neurons. After day 5, all neurons begin to degrade when cultured in L15 medium (see Section IV,B). In Medium 199, stage 3 neurons remain healthy for another 5-7 days, but cells that do not develop to stage 3 undergo gradual attrition. Bar: 22 ^m (A and B) and 44 ^m (C and D).

Indeed, the lack of technical and equipment needs for their most basic culture methodology even suits these neurons for use in undergraduate laboratory classes. With somewhat more refined protocols of culture, we have used them for toxicological assays (Heidemann et al., 2001) and for cell biological studies of axonal development (Chada et al., 1997). Having had experience culturing other neuronal cells, including chick sensory neurons, rat hippocampal neurons, and PC 12 cells, our goal is to draw attention to the substantial advantages of these neurons in terms of ease, animal-use issues, economy, productivity, and authentic neuronal character. That is, we would like to cordially invite the community of cell neurobiolgists to consider using and developing these neurons because we believe they would prove highly suitable for many applications of single cell neuronal culture. However, this will require additional studies to further optimize their development and to develop biochemical and genetic markers of the kind that exist for other cultured neurons. Although their culture was introduced in the mid-to-late 1970s by Sensenbrenner and colleagues, including a high-profile report (Pettman et al., 1979), only a few laboratories routinely used chick forebrain neurons in the 1980s and 1990s, notably that of A. Vernadakis in a wide variety of studies. We believe these neurons deserve wider use.

II. Growth and Development Characteristics

In addition to their technical conveniences, we chose to work with chick forebrain neurons because they share several useful growth characteristics with rat E19 hipppocampal neurons in low-density culture (Goslin and Banker, 1991). The latter is a standard preparation for a variety of cytological studies in which clear observations of neurites belonging to a particular cell body are needed, e.g., specification of axonal/dendritic polarity (Craig and Banker, 1994) and the role of the cytoskeleton in axonal function (e.g., Fischer et al., 1998). Unlike rat hippocampal neurons, however, chick forebrain neurons do not require "the black magic of nerve cell culture'' (Goslin and Banker, 1991) but grow well with simple procedures and conditions.

A. Chick Forebrain Neurons Develop into Typical Pyramidal Neurons in Low-Density Culture

We were initially attracted to chick forebrain neurons because they can be grown at low enough density to clearly identify neurites as axons belonging to a particular cell body, which was needed for cytomechanical work (Chada et al., 1997). We also found that these neurons develop their classical pyrimidal shape by a series of stereotyped stages. We used this property to quantitatively assess the inhibition of axonal morphogenesis by sublethal exposure to methylmercury (Heidemann et al., 2001), a potent neurotoxicant that is particularly damaging during early neural development (National Research Council, 2000). The stereotyped progression to axonal outgrowth is similar to that of rat hippocampal neurons as described by Dotti et al. (1988) from whose studies we have adopted our three stages of axonal development and our terminology. Fig. 1 shows the development of chick forebrain neurons during a 5-day period after plating. Shortly after plating (Fig. 1A), chick forebrain neurons show lamellipodial and filopodial motility all along their margin, as do hippocampal and most other neurons in culture. This early stage is called "stage 1.'' After a day in culture (Fig. 1B), most neurons have developed to "stage 2,'' which is characterized by short tapering neurites called "minor processes.'' Some cells remain in stage 2 and never develop axons. However, 50% or more of the cells extend a single (usually) axon and thereby develop to "stage 3'' by the third, fourth, and fifth day in culture (Figs. 1C and 1D). This axonal outgrowth occurs via rapid elongation of one of the minor processes, again very similar to axonal growth in hippocampal neurons [see Craig and Banker (1994) for a discussion of the development of axons from minor processes]. As shown in Figs. 1 and 2, individual neurons developing to stage 3 are typical pyrimidal brain neurons. This morphology not only allows for clear axonal identification, but is another advantage for student laboratory use because such pyrimidal neurons are the typical textbook illustration of a neuron.

B. Chick Forebrain Neurons Show Elongation Behaviors Similar to Hippocampal Neurons

Similar to hippocampal neurons (Ruthel and Banker, 1998, 1999), chick forebrain neurites often elongate via a cycle of retraction and subsequent net elongation accompanying the arrival of a "wave" of motility that propagates along the axon (Fig. 3). A motile growth cone-like structure appears near the proximal end of the neurite (Fig. 3A) and then moves distally toward the true growth cone (Fig. 3B). As the wave approaches the distal end, the neurite retracts (Fig. 3C), but then elongates again over the following 10-15 min (Fig. 3D) showing a net elongation of about the length of a growth cone. The motile behavior of the wave, retraction, and elongation behavior, as well as the time course and step size of elongation, are all very similar to rat hippocampal axons (see Fig. 2; Ruthel and Banker, 1999).

C. Chick Forebrain Neurons Differ in Important Ways from Hippocampal Neurons

The foregoing developmental, growth, and general shape similarities of hippocampal and chick forebrain neurons suggest to us that both cell types share certain properties that are probably conserved among embryonic forebrain pyrimidal neurons. There are also differences with hippocampal neurons in culture. Although one's first impression is the morphological similarity of the two neurons, direct comparison shows that chick forebrain neurons (Fig. 2A) have slightly larger, more rounded cell bodies and their neurites are a larger caliber than hippocampal neurons (Fig. 2B). Also, the sequence of development of chick forebrain neurons is slower by about a day compared to optimal cultures of hippocampal neurons. For our work, at least, these two differences proved useful insofar as larger cells are easier to manipulate and slower development allows for a

Chicken Embryonic Stem Cells

Fig. 2 Morphological comparison of chick forebrain neurons and embryonic rat hippocampal neurons in early stage 3. (A) Chick forebrain neurons show typical pyramidal morphology with a single axon, usually, and several nongrowing minor processes. These neurons have a particularly rounded cell body and are thus obscured by the halo that is characteristic of Zernike phase optics. (B) Rat hippocampal neurons also show a typical pyramidal shape, but both the cell body and the neurites are more delicate than those of chick forebrain neurons. Both neuronal types were cultured on polylysine-treated tissue culture plastic. Bar: 20 ^m.

Fig. 2 Morphological comparison of chick forebrain neurons and embryonic rat hippocampal neurons in early stage 3. (A) Chick forebrain neurons show typical pyramidal morphology with a single axon, usually, and several nongrowing minor processes. These neurons have a particularly rounded cell body and are thus obscured by the halo that is characteristic of Zernike phase optics. (B) Rat hippocampal neurons also show a typical pyramidal shape, but both the cell body and the neurites are more delicate than those of chick forebrain neurons. Both neuronal types were cultured on polylysine-treated tissue culture plastic. Bar: 20 ^m.

Chick Forebrain Neurons

Fig. 3 Motile growth cone-like waves accompany neurite elongation in chick forebrain neurons, as reported for hippocampal neurons. Frames are taken from analog time-lapse video recordings of a chick forebrain neuron in culture. (A) A motile wave (arrow) develops in the proximal region of a minor process. The lamellipodial activity of this thickening is clear in time lapse. (B) Three minutes later, this wave has moved distally along the neurite. (C) As the motility wave approaches the distal end of the neurite (4 min after B), the neurite retracts, which is followed some 8 min later (D) by net elongation and thickening of the neurite. These motility behaviors associated with outgrowth are similar in appearance, size, and time scale to those reported for rat hippocampal neurons by Ruthel and Banker (1999). These behaviors accompany approximately 40% of elongation bouts by chick forebrain neurons. Bar: 15 ^m.

Fig. 3 Motile growth cone-like waves accompany neurite elongation in chick forebrain neurons, as reported for hippocampal neurons. Frames are taken from analog time-lapse video recordings of a chick forebrain neuron in culture. (A) A motile wave (arrow) develops in the proximal region of a minor process. The lamellipodial activity of this thickening is clear in time lapse. (B) Three minutes later, this wave has moved distally along the neurite. (C) As the motility wave approaches the distal end of the neurite (4 min after B), the neurite retracts, which is followed some 8 min later (D) by net elongation and thickening of the neurite. These motility behaviors associated with outgrowth are similar in appearance, size, and time scale to those reported for rat hippocampal neurons by Ruthel and Banker (1999). These behaviors accompany approximately 40% of elongation bouts by chick forebrain neurons. Bar: 15 ^m.

longer time window to investigate initial axonal morphogenesis. Morphologically, at least, the minor processes of stage 3 chick forebrain neurons do not subsequently grow into well-developed dendrites and thus fail to develop to stages 4 and 5, as do hippocampal neurons (Dotti et al., 1988). Although chick forebrain neurons will grow at sufficiently low densities to count, manipulate, and image individual cells, they will not develop at the very low density reported for hippocampal neurons (1000 cells/cm2; Goslin and Banker, 1991). Plating efficiency and cell density results for chick forebrain neurons are discussed in Section IV,C.

III. Isolation of Single Chick Forebrain Neurons

The ease of locating, identifying, and dissecting the telencephalon of E8 chicks is one of the most attractive features of this preparation. Typical, brief trypsin treatment and dispersion or just simple mechanical disperson of this tissue layer produces a mixture of single cells and larger cell aggregates. A simple settling procedure then produces a reasonably uniform suspension of single neurons of 90-99% purity. A single embryo produces enough neurons for approximately fifty

60-mm plates at low density (300,000 cells/dish). In our hands, the entire procedure from cracking open the egg to a suspension of cells ready for plating takes less than 30 min, another feature that suits this culture system to undergraduate student laboratories.

A. Obtaining and Maintaining Chick Embryos

We obtain our chick embryos from our university's poultry farm. Workers at or near other "ag schools'' will likely enjoy the same convenient source, and others can use such academic poultry operations to obtain information about local poultry farms/hatcheries as suppliers of fertilized eggs and/or later embryos. Government agricultural agents are also a good source for such information. Fertilized chicken eggs are more widely available than later embryos because the former are produced for the organic food market. While fertilized, unincubated eggs in the retail store are not suitable for culture use, eggs from a local wholesale source usually are.

Conveniently, unincubated eggs (<24 h by the hen) can be stored at 15-18°C in an airtight food container for 7-10 days, which curtails embryonic development completely without damaging subsequent development (North and Bell, 1990). By placing these eggs into incubation every 1-2 days, a single purchase of fertilized eggs can provide 8-day-old embryos throughout a week. Longer term storage at lower temperatures progressively inhibits any subsequent development, which is presumably the reason refrigerated fertilized eggs from a retail store do not develop well. In contrast to unincubated eggs, eggs incubated by the hen for >24 h cannot be stored conveniently for useful lengths of time, although a reduced incubation temperature (34° C) will slow development.

The egg incubator we use to support normal development is an inexpensive styrofoam box with a fan and thermostatted heater that accepts the standard cardboard tray for 30 eggs (Hov-a-bator, G.Q.F. Mfg. Co, Savannah, Georgia; www.gqfmfg.com). At the bottom of this unit, there is a reservoir channel for water to humidify the eggs, which also should be turned every day. These or similar units can often be purchased from a local agricultural supply house (''feed-and-seed store'').

As the foregoing suggests, chick embryos are obtained and used under agricultural guidelines and practices. Their price reflects the economy of an agricultural commodity, and chicken eggs raise fewer animal-use issues than mammalian embryos. In cases where forebrain neurons can substitute for mammalian neurons, the use of chick embryos fulfills an ethical desire to substitute simpler animals for more complex in studies of primary neurons in culture.

B. Dissection of the Forebrain

In addition to standard tissue culture items, such as serological pipettes and sterile tubes, the only tools we use for dissection include two pairs of watchmakers forceps (Dumont #5, inexpensive "electronic grade'' suffice) and a sterile Pasteur pipette that has been scored and broken off to produce a 3- to 5-mm orifice and then fire polished and sterilized by a gas flame. Anesthesia of the embryo at E8 is impractical: the embryo begins to die after cracking the egg because rupture of the extraembryonic vasculature causes blood pressure to fall to near zero. The egg is cracked open into the top of a 100-mm petri plate, and the embryo is isolated with the forceps from the yolk and extraembryonic tissue. The embryo is decapitated quickly as near to the trunk as possible to allow the head to be immobilized on a wax-filled dissection plate by a pin through the neck. (We make our dissection dishes by filling a petri plate with melted histological embedding wax and allowing the wax to harden.) All waste is deposited into a plastic bag and disposed of as for food.

For dissection, magnification (from simple reading glasses to a dissecting scope) is helpful, but not absolutely required. The two forebrain hemispheres form an easily observed triangle of grayish tissue at the most forward portion of the head: one point is located just behind the beak; two sides of the triangle are next to each of the large eyes. The third side is formed by the left-to-right brain fissure separating the two forebrain hemispheres from the midbrain, which is seen easily by the vasculature running through this fissure. The neural tissue is overlain by two, thin almost transparent layers (later to become skin and bone) that should be removed individually with watchmakers forceps. All of the tissue of the embryo is quite soft at this early stage and both layers can be removed as if one were tearing wet tissue paper with forceps. It is helpful to begin the dissection at the central fissure of the forebrain so that the forceps pierce the two overlying layers in a gap between neural tissues, thus minimizing the risk of a stab wound to the brain hemispheres. Once the overlying tissue is removed, each telencephalon is removed by bringing open forceps down around and beneath the hemisphere. Subsequent squeezing and lifting of the forceps should release the hemisphere like removing a grape at the stem.

We process the isolated tissue through two 35-mm culture dishes containing Ca2+, Mg2+-free Hanks balanced saline (HBSS) buffered to pH 7.4 with 5 mM HEPES and containing 100 U/ml of penicillin G and 100 yg/ml streptomycin sulfate. In the first dish, we remove the overlying meninges, some of which may have been removed by previous dissection maneuvers. This very thin layer of cells on the outside (pial surface) of the hemispheres is highly vacularized and can be seen by its red tinge compared to the nearly white neural tissue. Complete and careful removal of the meninges ("never a trace of red'') is the most important step in ensuring cultures of nearly pure neurons. After removal of the meninges, the tissue is moved to the second dish of HBSS and minced with the forceps. Up until this point in the procedure, we have sterilized the forceps several times by a brief rinsing in alchohol and brief flaming. The tissue fragments are harvested from the dish by the broken Pasteur pipette. By allowing the tissue fragments to settle in this pipette, a drop or two will place all of the tissue at the bottom of a sterile 15-ml tube for dissociation.

C. Dissociation of Forebrain Tissue

Tissue dissociation is performed in HBSS. The simplest method is to vigorously vortex the tube containing the tissue fragments in 2 ml of HBSS and then gently aspirate through a 10-ml pipette. However, we routinely use a typical trypsin dissociation. We add 2 ml of 0.1% crude trypsin ("1:250 trypsin,'' Sigma) in HBSS to the tube containing the brain fragments and incubate in a 37°C water bath with vortexing every 2 min for about 8 min. After 8 min, the tissue is aspirated five or six times through a sterile 10-ml serological pipette and is then incubated another 2-5 min with occasional vortexing. Some tissue fragments usually remain and we let these settle to the bottom of the tube for a few minutes. Then, using a sterile Pasteur pipette, we transfer the supernatant suspension of single cells into another sterile tube. Simple swirling of this tube allows quite uniform aliquots of cells to be delivered from this tube into each culture vessel.

Not surprisingly, all cultures have some level of detritus and dead or abnormal cells. Unfortunately, some of this contamination is not removed easily because it sticks to the polylysine-treated culture surface (see later). However, living cells in the culture are nearly pure neurons, as claimed originally by Pettman et al. (1979), which is consistent with the finding that glia do not develop in the chick forebrain until E10 (Tsai et al., 1981). After dissociation and settling, we count the number of cells in a hemocytometer and find that we obtain 2-5 x 107 neurons of 90-95% viability (as shown by trypan blue exclusion) from the two hemispheres of a single embryo.

IV. Culture Conditions for Chick Forebrain Neurons

Chick forebrain neurons are grown on polylysine-treated culture surfaces, either glass or plastic. Like all other neurons we have cultured, chick forebrain neurons grow better on tissue culture plastic than on equivalently treated glass. We find that two "classic" culture media are particularly suitable for these neurons: Medium 199 is CO2 dependent and Leibovitz's L-15 is CO2 independent. We routinely supplement both with a variety of growth promoters, as discussed later. Like most brain neurons, chick forebrain neurons grow better at higher density, but good development and good single cell access are achieved at a plating density of 1 x 104 cells/cm2. In our hands, two widely used methods of improving the survival and development of brain neurons have in fact proven inhibitory to forebrain neurons: high K+ in the medium and coculture with glial-derived factors.

A. Polylysine Treatment of Tissue Culture Surfaces

Five to 10 min prior to the dissection of the embryo, we begin polylysine treatment of the culture surfaces. For plastic tissue culture surfaces, our protocol is to flood the surface with 0.1% poly-L-lysine hydrobromide (1 mg/ml, Sigma) in Dulbecco's phosphate-buffered saline (PBS) containing Ca2+ and Mg2+. Many laboratories use polylysine dissolved in 0.1 M sodium borate buffer, pH 8.5, which works equally well. We pipette off most of the solution, leaving only a thin layer of solution on the surface. The recovered solution is then used to flood the next surface, and so on. At the earliest opportunity we cover the culture vessel. Near the end of the trypsin dissociation outlined earlier, we rinse the culture surface three times in sterile H2O. Typically, the polylysine solution is in contact with the dish for approximately 30 min. At least on tissue culture plastic, longer treatment times do not improve attachment or development of these neurons.

For glass coverslips, we sterilize and clean ordinary glass coverslips by soaking them in absolute ethanol for 10 min. Using Dumont #5 forceps, we drain each coverslip and then ignite the ethanol by passing through a gas flame. (A high rate of coverslip cracking is usually due to insufficient drainage of the ethanol prior to ignition.) Polylysine treatment is similar to that described earlier except that rather than flooding the surface, we place approximately 1 ml of the same polylysine solution onto the coverslip. With care, the solution remains restricted to the coverslip by surface tension.

B. Culture Media

In terms both of longevity of culture and of the fraction of cells developing axons to stage 3, our best results have been obtained in Medium 199 (Table I), which was originally developed to support chick embryo fibroblasts (Morgan et al., 1955). This medium is superior to other CO2-dependent media we tried, including MCDB, RPMI 1640, Hams F12, DMEM, MEM, and neurobasal medium (Life Technologies Inc.). When using Medium 199, we equilibrate the medium in a 5% CO2 atmosphere prior to plating because chick forebrain neurons (like hippocampal neurons) are quickly damaged by exposure to alkaline conditions.

We also routinely use Leibovitz's L15 medium, which is buffered by its constituent amino acids, not CO2-bicarbonate, because our work has often involved extended periods of manipulation on a microscope stage in the ambient atmosphere (Chada et al., 1997). This medium supports slightly less robust axonal

Table I

Fraction of Chick Forebrain Cells Developing an Axon (Stage 3)

Table I

Fraction of Chick Forebrain Cells Developing an Axon (Stage 3)

Media tested

Third day of growth

Fifth day of growth

199 (n = 8)a

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