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.
Solutions
Day 1: Dissection of Aplysia abdominal ganglion
Preparation of the enzymatic dissociation solution
Dissection of abdominal ganglion
Day 2: Plating of Aplysia bag cells
Preparing poly-lysine-coated coverslips
Dissociation of bag cells
Figure 1. Schematic of Aplysia californica abdominal ganglion. Color lines indicate where to cut to obtain bag cell clusters.
Day 3: Setting up chamber for live imaging of Aplysia bag cells
Assembly of imaging chamber
Bag cells on the microscope
Since Aplysia bag cell neuronal cultures are at low density, it is best if your microscope setup has stage positioning functions to facilitate switching between cells. Bag cells can be easily kept at RT for several hours on stage during imaging experiments. Exchange the medium periodically to avoid evaporation.
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 recently released Aplysia genome information as well as improved expression techniques make these neurons a powerful model system for studying molecular and cellular mechanisms of neuronal growth cone motility and guidance.
We would like to thank Ryan Maneri (Oystercatcher Productions, LLC) for filming our procedure and Rodney McPhail (Department of Biological Sciences, Purdue University) for assistance with editing the dissection video. Research in the Suter lab is supported by grants from the NIH (R01 NS049233) and the Bindley Bioscience Center at Purdue University to D.M.S.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
#1 glass coverslips 22×22 mm | Tool | VWR | 48366067 | |
#1.5 glass coverslips 22×22 mm | Tool | Corning | 2870-22 | |
35 mm Petri dishes | Tool | Falcon | 353001 | |
Filters for medium filtration | Tool | 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 | C79-500 | |
Gentamicin | Reagent | Invitrogen | 15750-060 | |
HEPES | Reagent | Sigma | H4030 | |
L15 medium | Reagent | Invitrogen | 41300-039 | |
L-glutamine | Reagent | Sigma | G8540 | |
Magnesium chloride | Reagent | Mallinckrodt | 5958-04 | |
Magnesium sulfate | Reagent | Mallinckrodt | 6070-12 | |
Poly-L-lysine (70-150 kD) | Reagent | Sigma | P6282 | |
Sodium chloride | Reagent | Mallinckrodt | 7581 | |
Neutral Protease (Dispase) | Reagent | Worthington | LS02111 |