Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Summary

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Abstract

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Protocol

Solutions

• L15-ASW cell culture medium (1l)
  • 1 bag of L15 powder
  • add 800 ml of H₂O ultrapure
  • NaCl 400 mM
  • MgSO₄         27 mM
  • MgCl₂          28 mM
  • L-Glutamine   4 mM
  • Gentamicin    50 µg/ml
  • HEPES         5 mM
  • Adjust to pH 7.9
  • Add drop by drop CaCl₂ 9.3 mM (stop if precipitates)
  • Add H₂O ultrapure to 1 l
  • Check osmolarity (950-1000 mmol/kg) with osmometer (vapor pressure, Wescor # 5520)
  • Filter 0.22 µm using a positive pressure filtration unit (Filters: Millipore SVGV010RS)
  • Store at 4....

Discussion

Aplysia bag cell neurons provide a serum-free neuronal cell culture system with very few non-neuronal cells. These neurons form very large growth cones suitable to address a number of important cell biological questions. Bag cell neurons can easily be manipulated and imaged at room temperature over several hours. Using Fluorescent Speckle Microscopy (FSM) one can quantitatively analyze the various parameters of F-actin and microtubule polymerization and translocation dynamics. These imaging tools together with the recent.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#1 glass coverslips 22x22 mm	Tool	VWR international	48366067
#1.5 glass coverslips 22x22 mm	Tool	Corning	2870-22
35 mm Petri dishes	Tool	Falcon BD	353001
Filters for medium filtration	Tool	EMD Millipore	SVGV010RS
High vacuum grease	Tool	Dow Corning	1597418
Osmometer	Tool	Wescor	5520
Plastic shims	Tool	Small Parts, Inc.	SHSP-200
Calcium chloride	Reagent	Fisher Scientific	C79-500
Gentamicin	Reagent	Invitrogen	15750-060
HEPES	Reagent	Sigma-Aldrich	H4030
L15 medium	Reagent	Invitrogen	41300-039
L-glutamine	Reagent	Sigma-Aldrich	G8540
Magnesium chloride	Reagent	Mallinckrodt Baker Inc.	5958-04
Magnesium sulfate	Reagent	Mallinckrodt Baker Inc.	6070-12
Poly-L-lysine (70-150 kD)	Reagent	Sigma-Aldrich	P6282
Sodium chloride	Reagent	Mallinckrodt Baker Inc.	7581
Neutral Protease (Dispase)	Reagent	Worthington Biochemical	LS02111