Dissection and Culturing of Chick Ciliary Ganglion Neurons A System Well Suited to Synaptic Study

Barbara W. Bernstein

Department of Biochemistry and Molecular Biology and

Program in Molecular, Cellular, and Integrative Neurosciences Colorado State University Fort Collins, Colorado 80523

I. General Introduction

A. Neuroanatomy and Development of the Ciliary Ganglion

B. Applications of Cultured Cells and Isolated Calyx Terminals II. Materials

A. Materials for Dissection

B. Materials for Dissociation and Culturing

III. Methods

A. Dissection: Isolation of Ciliary Ganglion

B. Dissociation of the Ganglion

C. Culturing of Ciliary Neurons

IV. Variations on Dissection, Dissociation, and Culturing Themes V. Concluding Comments

References

This chapter describes the function and development of the ciliary ganglion, the potential of ciliary ganglion neurons as a cell biological tool, and their dissection, dissociation, and culturing. Ciliary ganglion neurons grow unusually rapidly on a laminin-based substratum and develop large, thin calyx terminals in culture in less than 12 h. The two neuronal classes present in the cultures can be identified by size alone. The limited number of ganglia per animal renders this ganglion a poor choice for biochemical studies based on the extraction of cultured cells. However, they are ideally suited for studies based on single-cell observation, particularly investigation of presynaptic mechanisms using fluorescence microscopy.

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

I. General Introduction

Like all other neuronal cell culture preparations, the ciliary ganglion has distinct advantages and disadvantages that must be weighed according to the research needs. One of its primary advantages is the relative homogeneity of its neurons, which is typical of parasympathetic ganglia because of their limited number of targets. There are only two neuronal types, ciliary and choroid, both of which are cholinergic and receive cholinergic input (Marwitt et al., 1971; Chiappinelli and Dryer, 1984).

A. Neuroanatomy and Development of the Ciliary Ganglion

The ganglion lies in the caudal part of the orbital cavity at the junction of the lateral side of the optic nerve and the extraoccular muscle, sending a short branch to the occulomotor nerve. Ciliary cells receive midbrain input through single, large calyciform terminals (Martin and Pilar, 1963; Dryer and Chiappinelli, 1987), whereas choroid cells receive multiple small boutons. Ciliary cells control light reflex and visual accommodation through the innervation of striated muscle in the iris and ciliary body, whereas choroid neurons, distinguished easily by their smaller somata diameter (13 ± 2 ^m compared to 23 ± 3 ^m of ciliary neurons; McNerney et al., 2000), innervate the smooth muscle of choroid coat blood vessels. If a more stringent identifying criterion of ciliary neurons is required, they can be labeled by incubating the ciliary nerves of the intact ganglion in the fluorescent dye Dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per-chlorate; Haugland, 1996; McNerney et al., 2000). These two neuron classes both have (aBgt)-AChRs, composed of a7 subunits and inhibited by bungarotoxin, and a3 -AChRs, composed of a3, a5, and ^4 subunits and insensitive to bungar-otoxin, but choroid and ciliary neurons have distinctively different electrophysio-logical characteristics (McNerney et al., 2000).

B. Applications of Cultured Cells and Isolated Calyx Terminals

This avian ganglion is a favorite model system for the study of embryonic development (Dryer, 1994; Nishi, 1994) and neurotrophic signaling (Finn and Nishi, 1996; Ip and Yancopoulos, 1992) because of the ease of manipulating target tissue and inputs (accessory part of the occulomotor nucleus; Lee et al., 2001). They undergo a fairly complex pattern of cell death that is coordinated with synapse formation, involves replacement by postmitotic precursor cells, and is target driven rather than programmed (Barde, 1989). Neuronal degeneration starts as early as HH stage 24, i.e., before embryonic day 5 (Lee et al., 2001; Hamburger and Hamilton, 1951).

Investigators have taken advantage of the high efficiency (Borasio et al., 1989; Yamashita et al., 1999) with which proteins can be trituration loaded into these neurons before plating. The efficiency of loading untagged species can be

Fig. 1 Forty percent of the neurons were loaded through trituration. The fluorescence image of cells containing rhodamine-tagged cofilin, a protein that regulates actin filament turnover (Bamburg, 1999), was merged with a phase image of the same field. Cells were grown overnight on a glass coverslip etched with a lettered matrix (trade name Cellocate; Brinkmann, Westbury, NY). The matrix allows one to identify and image the same cell area repeatedly even after returning the culture to the incubator. Bar: 175 ^m. (See Color Insert.)

Fig. 1 Forty percent of the neurons were loaded through trituration. The fluorescence image of cells containing rhodamine-tagged cofilin, a protein that regulates actin filament turnover (Bamburg, 1999), was merged with a phase image of the same field. Cells were grown overnight on a glass coverslip etched with a lettered matrix (trade name Cellocate; Brinkmann, Westbury, NY). The matrix allows one to identify and image the same cell area repeatedly even after returning the culture to the incubator. Bar: 175 ^m. (See Color Insert.)

monitored with FITC-dextran. In the first study, isotope-tagged RhoA (5 mg/ml) or C3 transferase (0.1 mg/ml) was used to reveal the linkage between p75ntr and actin assembly in neurite outgrowth. Fluorescently tagged cytoskeletal proteins (see Fig. 1), chromophore-tagged antibodies for CALI studies (chromophore-assisted laser inactivation; Beermann and Jay, 1994), and transfecting DNA plasmids can also be trituration loaded efficiently. Introduction of proteins via microinjection should be feasible, as the large soma has a relatively small nucleus compared to many neurons in which the nucleus occupies the entire cell body (see Fig. 2). The large flat, thin calyx terminal formed between ciliary neurons in culture (Fujii and Berg, 1987) provides an ideal structure for presynaptic terminal research with fluorescence microscopy (see Fig. 3). The calyx terminal fits like a bathing cap over the postsynaptic cell body. It can be specifically loaded with a tagged protein via a sequence of hyperosmotic and hypoosmotic medium incubations (see Fig. 4). Instructions and materials for this protocol can be obtained as a kit from Molecular Probes (Influx; Eugene, OR).

Alternatively, the calyx terminal has been studied immediately after cell dissociation as an isolated pre- and postsynaptic structure, i.e., a terminal with an attached cell body, removed intact from the ganglion. This structure can be isolated with a combination of proteolytic enzymes (collagenase, hyaluronidase, dispase; Stanley and Goping, 1991) with more limited activity than trypsin, which is used commonly for ganglion dissociation. This proteolytic combination digests glia/neuron connections more effectively than neuron/neuron connections. The

Fig. 2 Ciliary neurons have a relatively small nucleus that occupies ~25% of the somata area. Images, showing fluorescent DAPI staining of the nucleus, were overlayed on phase images of the same field.

(A) Overnight culture. Bar: 10 ^m. (B) Three-hour culture. Bar: 20 ^m. (C) One-hour culture. Bar: 15 ^m. (See Color Insert.)

Fig. 3 Active calyx presynaptic terminals are visualized with the fluorescent styryl dye FM1-43 (Cochilla et al, 1999). Cells were incubated in 10 ^M FM1-43 and were depolarized with an isotonic 75 mM K+ buffer. FM1-43 is internalized in terminals only when they are depolarized, and neurotransmitter vesicles are formed through endocytosis. (Right) Overlay of a FM1-43 image (center) and phase image (left). Bar: 20 ^m. (See Color Insert.)

Fig. 4 Membrane-impermeable molecules can be loaded into terminals via Influx (Molecular Probes). A schematic of Influx modified from the "Molecular Probes Handbook'' illustrates the mechanism of loading species through incubation first in hypertonic medium containing impermeable molecules (A), which are taken up in endocytotic vesicles that are then lysed by switching the culture to hypotonic medium (B). FM1-43, loaded by depolarization (C), identifies active terminals, which are the same structures loaded with Texas red-tagged dextran (20 kDa) via the Influx kit (D). Bar: 20 ^m. (See Color Insert.)

Fig. 4 Membrane-impermeable molecules can be loaded into terminals via Influx (Molecular Probes). A schematic of Influx modified from the "Molecular Probes Handbook'' illustrates the mechanism of loading species through incubation first in hypertonic medium containing impermeable molecules (A), which are taken up in endocytotic vesicles that are then lysed by switching the culture to hypotonic medium (B). FM1-43, loaded by depolarization (C), identifies active terminals, which are the same structures loaded with Texas red-tagged dextran (20 kDa) via the Influx kit (D). Bar: 20 ^m. (See Color Insert.)

neuromuscular junction in vitro is yet another area of investigation for which this preparation is appropriate (Campagna et al., 1997; Bruses et al., 1995).

A major disadvantage of ciliary ganglia for cell culture studies is the small number of neurons obtainable per animal. There are 17,500/ganglion at embryonic day 8 (E8; see Fig. 5; Lee et al., 2001). Only 2 ganglia are present per animal

Fig. 5 Time course of neuronal proliferation and degeneration (data from Lee et al, 2001). Data represent the mean of 8-11 ganglia ± SEM.

Embryonic age, days

Fig. 5 Time course of neuronal proliferation and degeneration (data from Lee et al, 2001). Data represent the mean of 8-11 ganglia ± SEM.

as opposed to 10-30 ganglia for other peripheral nervous system tissue, such as sympathetic or dorsal root ganglia.

II. Materials

A. Materials for Dissection

Dissecting microscope with illumination from above (e.g., Nikon SMZ-U 1:10 zoom)

Egg incubator

High-intensity lamp for candling eggs (blood vessel network visible in a healthy, well-developed egg) Laminar flow hood Scalpel

Two pairs of Dumont No. 5 forceps

Crocus cloth, grind stone (sharpening and polishing forceps) Ethanol

~4-in. square dish filled with Sylgard (a silicone plastic; Dow Corning, Midland, MI)

Plastic culture dishes (Falcon; 35, 60, or 100 mm diameter)

B. Materials for Dissociation and Culturing

Trypsin (Sigma-Aldrich, St. Louis, MO; should be kept in frozen aliquots to avoid repeated freeze/thaw cycles)

Matrigel (Becton-Dickinson Labware, Bedford, MA; frozen 11 mg/ml stock) Penicillin/streptomycin (Sigma-Aldrich)

Neurobasal medium (Invitrogen, Carlsbad, CA; Brewer et al., 1993)

B27 supplement (Invitrogen)

Glutamine (200 mM frozen stock; Sigma-Aldrich)

Hank's balanced salt solution (HBSS; Sigma-Aldrich)

Humidified CO2 tissue culture incubator

No. 1 glass coverslips

A. Dissection: Isolation of Ciliary Ganglion

For ease of handling, we have chosen E10 ganglia, which have close to the maximum number of neurons (Fig. 5; Lee et al., 2001). However, by E10 there are more nonneuronal cells than at E8. Before starting, all materials are sterilized by autoclaving, rinsing with 70% ethanol, or exposing to ultraviolet (UV) light for >20 min. If culturing cells for <12 h, sterilization is not critical.

1. When growing cells for more than 2 days, precoating No. 1 glass coverslips with 100 yg/ml poly-D-lysine is recommended to prevent cell clumping. Store poly-D-lysine at —20°C (5 mg/ml). Dilute it 50x in borate buffer [1.24 g boric acid and 1.90 g sodium tetraborate (borax) in 400 ml H2O]. Apply 100-150 yl/ coverslip (^130 mm2), incubate for 30 min, remove poly-D-lysine, wash 3x ultrapure H2O, air dry, UV sterilize (20 min), and use coverslips within a week.

2. Warm to room temperature trypsin, Dulbecco's modified Eagle's medium (DMEM)/10% fetal bovine serum (FBS), and HBSS without Ca2+ or Mg2+; trypsin is inhibited by Ca2+. Warm to 37°C complete neurobasal growth medium (neurobasal medium/1:50 dilution B27 supplement/2 mM glutamine/100 U/ml penicillin/100 yg/ml streptomycin).

3. Apply enough Matrigel (100 yg/ml) to No. 1 glass coverslips to cover the area on which cells will be grown, i.e., Matrigel volume approximately equal to the volume of the cell suspension to be plated. Coverslips may be sealed permanently to the bottom of plastic drilled-out dishes or placed in any sterile plastic container (square or round dish of suitable size). Incubate coverslips with Matrigel for 30 min to several hours in a humidified 37°C/5% CO2 incubator while preparing cells. Do not allow Matrigel to dry.

4. Ganglia can be collected in a 500-yl microfuge tube containing 50 yl HBSS.

5. Spray egg shell with 70% ethanol. Use the blunt end of forceps to tap through the shell. Repeat tapping to form a circular crack that approximates the air sac perimeter. Use forceps to lift the cracked top off the egg. Gently lift chick from the egg, holding below the head with forceps.

6. Place the whole embryo on a dish filled with black Sylgard (a rubbery material). Surgically decapitate chick with a scalpel. Discard body. Pin the head to Sylgard with beak up. Pins the size of standard sewing pins are appropriate. Push one through the lower neck and one through each eye at an angle away from the dissector and toward the dish sides (see Fig. 6.1). The drawn white circle in Fig. 6.1 indicates the area of eyeball completely covered by tissue that must be removed. Bright white blotches on panels are reflected light from well-wetted surfaces. They can be eliminated by adjusting the polarizing filter on the microscope; unfortunately, this filtering reduces the illumination intensity.

7. Completely peel away the lower beak and then the upper beak (see Fig. 6.2). This can be done under the lowest magnification of a dissecting microscope.

8. Remove connective tissue covering the exposed eye. Try to remove as large a sheet of tissue as possible, which includes the caudal region of the eye. Be careful not to puncture the eyeball. If the eyeball collapses or pigment starts flaking off the eye, it is much more difficult to find the ganglion (see Figs. 6.3-6.9). The drawn white circle in Fig. 6.9 indicates the area of the eye from which the covering tissue has been removed.

9. Remove as much connective tissue as possible from over the extraoccular muscles without inadvertently removing the ganglion. Reposition the eye pin so the head of the pin is closer to the bottom of the dish. This can be done by merely loosening the pin attachment to the dish without removing the pin from the eye. The repositioning rotates the dorsal surface of the eye upward, often exposing the ganglion if sufficient connective tissue has been removed. It may help to rinse the intraoccular space with HBSS, which removes fragments of connective tissue and floats apart layers of tissues (Fig. 6.6). Increasing microscope magnification slightly is sometimes helpful.

10. The circled area in Fig. 6.10 is the ''7 o'clock'' position of the circle in Fig. 6.9. Use one pair of forceps to lift the ganglion from the area encircled in Fig. 6.10. The ganglion is located in the "V" formed by the optic nerve and the extraoccular muscles. Lifting the ganglion exposes its nerve trunks. The trunks and slightly greater optical density of the ganglion (circled in Fig. 6.17) distinguish it from abundant small chunks of connective tissue (see Figs. 6.11-6.17). Use the other pair of forceps to pluck out the ganglion without crushing it. A fully extracted ganglion is seen at the tip of the forceps (Fig. 6.18) with completely retracted

Fig. 6 Dissection of a left ciliary ganglion from a 13-day chick embryo. Images were captured with Metamorph software (Universal Imaging Corp., West Chester, PA) from a VCR tape recording of the dissection viewed through a dissecting microscope fitted with a DAGE camera. Magnification is the same in all panels. (1) The circled area indicates tissue covering the eyeball through which a pin is positioned. (2) Focus is shifted down to the lower beak, which is removed along with the upper beak. (3-9) Tissue is removed from the eyeball surface. (9) The circle shows cleared surface. (10) The small circled area, positioned at ''7 o'clock'' on the circle (9), contains a barely visible ganglion, which can often be seen after repositioning the pin through the eye. (11-18) Removal of the ganglion. (17) The circled area is the lifted ganglion attached by only one nerve trunk.

trunks, which make it appear twice as big as in previous panels. If necessary, dip the forceps tip up and down through the HBSS meniscus to wash the ganglion from the tip.

11. The ganglion can be kept at room temperature in HBSS for some time while collecting more. When all are collected (e.g., four ganglia), they can be trypsinized together.

B. Dissociation of the Ganglion

1. Add 10-15 yl 2.5% trypsin to 50 yl HBSS containing ganglia. Incubate for 10-15 min in a 37°C/5% CO2 incubator (described by Collins and Dawson, 1982).

2. Add 500 yl DMEM/10% FBS to inhibit trypsin. Let the ganglia settle to the bottom of the tube before removing all but ~10 yl of the medium.

3. Add ^100 yl growth medium: neurobasal medium, 1/50 dilution B27 supplement, 2mM glutamine, 100 U/ml penicillin, and 100 yg/ml streptomycin. With ganglia on the bottom of tube and at the lowest magnification, adjust the microscope light and focus to visualize ganglia. Draw ganglia repeatedly up and down through the opening of a 200-yl pipette tip (yellow tip) or a fire-polished glass Pasteur pipette held close to the bottom of the tube. Ganglia fall apart, often leaving a piece of undigested capsule; dissociated cells give the solution a granular appearance. It should require 30-70 cycles of repetitive pipetting to dissociate the cells. If fewer cycles are required, it suggests that the enzymatic treatment is too strong to maximize cell survival.

4. Adjust the growth medium volume to plate the cells at a desired density on the coverslip. For example, 1/5-1/10 ganglion may be plated in 50 yl and allowed to adhere to the substratum for 15-30 min in the incubator before bringing the final volume of medium in a 35-mm plastic culture dish to 2 ml.

C. Culturing of Ciliary Neurons

Immediately after dissociating ganglia, remove coverslips from the incubator, withdraw Matrigel, and plate the cell suspension without allowing the Matrigel residue to dry. We have grown cells for 48 h at a cell density as low as 1/16 ganglion/13 mm2 (area covered by 50 yl of medium); we have counted 7.4 x 104 cells/ganglion of which 1.6 x 104 are neurons (Lee et al., 2001). After this time, cells that were originally well isolated form large clusters, which for many purposes is unacceptable (see Fig. 7A). Cell aggregation can be prevented by choosing a more tightly adhering substratum than Matrigel, such as poly-D-lysine or a combination of poly-D-lysine and Matrigel. Matrigel is a solubilized basement membrane from a mouse sarcoma; it is predominantly laminin with some collagen and heparin sulfate proteoglycans and lesser concentrations of entactin, nidogen, transforming growth factor fl, fibroblast growth factor (FGF) and other growth factors.

Fig. 7 Ciliary neurons cluster when cultured for 2 days on Matrigel alone (A). This clustering can be avoided by first coating coverslips with poly-D-lysine and then applying Matrigel (B). Bar: 20 ^m.

Many possibilities exist for handling coverslips when doing live cell microscopic imaging. Coverslips are available as circles, squares, or rectangles in sizes ranging from 12 mm2 to 22 x 70 mm. They can be sealed permanently with 100% silicone aquarium sealant (All-Glass Aquarium Co., Franklin, WI) to drilled-out dishes. To avoid chemical toxicity, sealing should be done the day before plating cells to allow 24 h for the sealant to cure. Alternatively, coverslips can be left floating freely in dishes filled with medium and sealed reversibly to the bottom of drilled-out dishes immediately before transfer to a microscope stage; one can rim the opening of a drilled dish with Dow Corning high vacuum grease and press the dish over the coverslip with growing cells. Silicone gaskets, with or without attached clear plastic covers (Grace Bio-Labs, Bend, OR), can be sealed to dry coverslips before plating. These gaskets restrict the area of cell growth and can be used to limit the volume of incubating solution to <70 ^l. Small culture volumes may be important if reagents are limiting or expensive. It is not recommended to use such small volumes for long-term culture. Neuronal growth is preferred with a medium depth of >5 mm (unpublished data; mechanism currently under investigation).

IV. Variations on Dissection, Dissociation, and Culturing Themes

Laminin (Collaborative Biomedical, Bedford, MA) combined with poly-D-ornithine, poly-DL-ornithine (Bruses et al., 1995), or poly-D-lysine sustains cultures longer term. These cells have been kept for up to 2 weeks (Chen et al., 2001) when changing the medium every 2-3 days: minimum essential medium (MEM) with penicillin/streptomycin, 2 mM glutamine, 10% (v/v) heat-inactivated horse serum, and 3% embryonic chick eye extract (Nishi and Berg, 1981). Ciliary ganglion neurons have been cultured under a variety of conditions other than Matrigel alone. They include the following.

a. Poly-D-lysine and laminin coverslips with recombinant ciliary neurotrophic factor ([his]6-chCNTFyyy 10 ng/ml) (Lee et al, 2001).

b. Coverslips coated sequentially with poly-L-ornithine and laminin (Becton-Dickinson Labware) and Ham's F-12 medium (Becton-Dickinson Labware) supplemented with 10% fetal calf serum; 50 ng of combined nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 (Sigma-Aldrich) (Yamashita et al., 1999). The developmental time course of responsiveness to NGF and other neurotrophins must be considered; for NGF it peaks at E8 (Collins, 1988).

c. Coverslips coated sequentially with poly-D-lysine and Matrigel (see Fig. 7B).

Ciliary neuronal cells from E8 to E14 chick have been cultured. The dissection of chicks at different stages is qualitatively different due largely to the increase in connective tissue and cartilage with age. Dissection of E8 ganglia involves a totally different approach, one in which the eyeball is partially released from the socket (Nishi, 1996).

Dissociation has been accomplished with trypsin concentrations as low as 0.05% with incubation times extended to 20 min or more and ganglia first hemi-sected with a scalpel, thus circumventing the capsule. Collagenase, a milder and more specific protease than trypsin, has also been used (Chiappinelli et al., 1981).

Additionally, when necessary to achieve a particular research goal, intact ganglia can be maintained in complete neurobasal medium (Section III,A,1) in a humidified CO2 incubator for 24 h before dissociating and plating the cells. An example of a situation for which the maintenance of intact ganglia is convenient is the adenoviral infection of neurons for exogenous gene expression. One can dissociate ciliary cells immediately after dissection and culture them for 24 h before infecting with adenovirus containing a green fluorescent fusion protein (see Chapter 19). Alternatively, one can hemisect the ganglia with a scalpel to increase the exposed surface area of neurons and infect overnight with the virus before dissociating and plating. A similar holding procedure has been used to label ciliary cells with bungarotoxin (Bruses et al., 2001).

For some purposes, the enrichment of neurons over glia and fibroblasts is necessary. Removal of nerves and mesenchymal tissue before enzymatic incubation helps in this regard. In this case, ganglia should be collected in a small culture dish rather than in a microfuge tube. Nerve and connective tissue are teased away from the ganglion with two forceps. Preplating of dissociated cells is an additional commonly followed procedure, although it reduces neuronal yield. Cells are plated for ~60 min at 37°C; then the neurons, which adhere more weakly than nonneuronal cells, can be shaken off and replated. One can also use horse serum, which is reported to be less mitogenic than fetal calf serum, and FGF, which does not support nonneuronal cell proliferation (Nishi, 1996). Low concentrations of cytosine arabinoside (<10~5 M) or 10~5 M fluorodeoxyuridine plus uridine have been recommended for inhibiting nonneuronal cell division while minimizing neuronal toxicity. A less commonly used protocol to enrich neurons involves centrifugation of the suspension on a discontinuous Percoll gradient (30/60%) for 20 min at 500 x g, 4°C and collection of the neurons at the interface (Bruses et al, 1995).

V. Concluding Comments

This chapter describes in some detail procedures for dissecting, dissociating, and culturing chick ciliary ganglion neurons in some detail. They are methods that we have found appropriate for our research interests. They are presented here merely as a starting point and should be modified according to individual needs. Similarly, while vendors of some materials have been named, these are not necessarily endorsements. In most cases, several suppliers are available.

If large quantities of protein are needed for biochemical studies of cell extracts, the limited number of ganglia per animal makes ciliary ganglion neurons a poor choice. If single cell study is appropriate, i.e., one is using electrophysiology or various forms of electron or light microscopy, then these cells are a good choice. They grow rapidly, are generally robust, are efficiently loaded with macromol-ecules by trituration, can be loaded with macromolecules via Influx (incubation in a sequence of hyperosmotic and hypoosmotic medium), and form axonal/somatic synapses in culture.

Acknowledgments

I thank Professor James R. Bamburg, in whose laboratory this work was performed, and the agencies who support his research: the Alzheimer's Association (Grant IIRG-01-2730) and National Institutes of Health (Grants GM 35126 and NS40371). Adenovirus was supplied by L.S. Minamide.

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