Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Summary

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

Abstract

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues¹ and survival factors² located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons³. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons⁴. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro^5,6.

Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth.

SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol⁷) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival⁸.

Protocol

Recipes

Chick Ringers

7.2 g	NaCl
0.23 g	CaCl2 + 2H2O
0.37 g	KCl
0.115 g	Na2HPO4
900 ml	Water (Tissue culture grade)

Note: Adjust pH to 7.4. Bring final volume to 1000 ml with water.

10X PBS

40 g	NaCl
1 g	KCl

Discussion

We present a method to dissect and culture E4 SAG and E6 spinal cord explants, from chick, under serum-free conditions. This procedure is currently used in our lab to study the effects of various secreted ligands on SAG neuron survival and neurite outgrowth. Novel aspects of this protocol include the use of a serum-free system for culturing explants and the use of beads soaked in growth factors to study effects on SAG neurite outgrowth. A bead method similar to ours has been described for the chick spinal cord¹⁰

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dissection microscope			We use a Leica M80 stereomicroscope with brightfield base illumination
Slide warmer	C.S. E.		Collagen polymerization
Chicken egg incubator
37°C/5% CO2 humidified cell culture incubator
Sylgard© coated petri dish
24-well cell culture plate	Corning	3526
#5 Dumont forceps	Fine Science Tools	11252-30
#55 Dumont forceps	Fine Science Tools	11295-51
Stainless steel dissecting pins	Fine Science Tools	26002-10	Embryo pinning, fine dissection
Moria perforated spoon	Fine Science Tools	10370-17	Embryo harvest/transfer
200 μl wide mouth pipet tips	Dot Scientific, Inc.	118-96R	Explant transfer
E4 and E6 chicken eggs
Chick Ringer’s solution			Embryo harvest
HBSS	Sigma-Aldrich	H8264	SAG dissection
L15 medium	Sigma-Aldrich	L5520	Spinal cord dissection medium
Fetal calf serum	Atlanta Biologicals	S11150	Spinal cord dissection medium
Vannas Scissors	World Precision Instruments, Inc.	501778	Spinal cord dissection
Rat-tail collagen Type I	BD Biosciences	354249	Explant culture
10X PBS (Sterile)			To neutralize collagen
1N NaOH (Sterile)			To neutralize collagen
Distilled water (Sterile)			To neutralize collagen
pH indicator papers (6.0-8.1)	Whatman, GE Healthcare	2629-990	To check collagen pH
15 ml conical tubes	Dot Scientific, Inc.	818-PG
500 μl wide mouth pipet tips	Dot Scientific, Inc.	119-R100	To pipet collagen
DMEM/F12 medium	Sigma-Aldrich	D8437	Explant culture medium
ITS+1	Sigma-Aldrich	I2521	Medium supplement
Penicillin-Streptomycin	Invitrogen	15140-122	Medium supplement
CNTF (rat)	Sigma-Aldrich	C3835	Medium supplement
NT-3 (human)	Sigma-Aldrich	N1905	Medium supplement
Purified mouse Wnt5a	R&D Systems	645-WN-010
Affi-gel Blue gel beads	Bio-Rad	153-7302
Paraformaldehyde	Sigma-Aldrich	P6148	Fixation
PBS			Rinses
Triton X-100	Sigma-Aldrich	T9284	Blocking solution
Sodium azide	Sigma-Aldrich	S8032	Blocking solution
Calf serum	Invitrogen	16170-078	Blocking solution
Mouse monoclonal IgG2b anti-β Tubulin 1+II antibody	Sigma-Aldrich	T8535	Labels cell bodies and neurites
Alexa fluor 488 goat anti -–mouse IgG2b antibody	Invitrogen	A21141	Detects β Tubulin antibody
Teflon micro spatula	VWR international	57949-033	Release collagen gels from well