186
Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 14
Snail (Pulmonata) Neurons
Materials
Equipment - Culture chambers (made by attaching a small glass ring (1 cm diameter, 5 mm height) to a glass cover slip (18 x 18 mm) using melted
vaseline. The culture chambers are treated with 0.5 ml of a 0.1 % aqueous solution of concanavalin A (Sigma) for 1 h at 15°C, and washed
three times with deionized water and once with culture medium. The
chambers are prepared on the day of the experiment)
- Scissors
- Forceps
- Watch glass
- Petri dishes (7 cm diameter)
Solutions - Culture medium (Leibovitz's L-15 medium + snail physiological saline solution (see Appendix 1) in a ratio of 1 : 1, supplemented with
10 mM HEPES and 50 mg/l gentamicin)
- 0.5% Pronase
Animals - Adult snail Helix pomatia
Miscellaneous - Parafilm (American National Can Co.)
Procedure
Dissection 1. Remove the circum esophageal ganglionic complex (Fig. 19).
2. Treat the ganglionic complex with 0.5% pronase added to the snail
physiological saline solution for 2-2.5 h at 34°C.
3. Wash the ganglionic complex with 80% L-15 medium for 2 h at 4°C.
4. Transfer the ganglionic rings into a sterile watch glass containing the
culture medium.
5. Cut the connective tissue sheath using fine scissors.
6. Divide the ganglionic complex into individual ganglia.
Dissociation 1. Dissociate into single cells by trituration and pipetting.
Culture 1. Transfer the isolated neurons (without any processes) into the small
set-up
glass chambers coated with concanavalin A and fill up with culture
medium, several of which are placed in a sterilized petri dish (7 cm
diameter).
2. Allow the cells to settle and adhere to the bottom of the culture chambers for 10 min.
3. Fill the space between culture chambers within a petri dish with culture medium.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 14
Snail (Pulmonata) Neurons
Materials
Equipment - Culture chambers (made by attaching a small glass ring (1 cm diameter, 5 mm height) to a glass cover slip (18 x 18 mm) using melted
vaseline. The culture chambers are treated with 0.5 ml of a 0.1 % aqueous solution of concanavalin A (Sigma) for 1 h at 15°C, and washed
three times with deionized water and once with culture medium. The
chambers are prepared on the day of the experiment)
- Scissors
- Forceps
- Watch glass
- Petri dishes (7 cm diameter)
Solutions - Culture medium (Leibovitz's L-15 medium + snail physiological saline solution (see Appendix 1) in a ratio of 1 : 1, supplemented with
10 mM HEPES and 50 mg/l gentamicin)
- 0.5% Pronase
Animals - Adult snail Helix pomatia
Miscellaneous - Parafilm (American National Can Co.)
Procedure
Dissection 1. Remove the circum esophageal ganglionic complex (Fig. 19).
2. Treat the ganglionic complex with 0.5% pronase added to the snail
physiological saline solution for 2-2.5 h at 34°C.
3. Wash the ganglionic complex with 80% L-15 medium for 2 h at 4°C.
4. Transfer the ganglionic rings into a sterile watch glass containing the
culture medium.
5. Cut the connective tissue sheath using fine scissors.
6. Divide the ganglionic complex into individual ganglia.
Dissociation 1. Dissociate into single cells by trituration and pipetting.
Culture 1. Transfer the isolated neurons (without any processes) into the small
set-up
glass chambers coated with concanavalin A and fill up with culture
medium, several of which are placed in a sterilized petri dish (7 cm
diameter).
2. Allow the cells to settle and adhere to the bottom of the culture chambers for 10 min.
3. Fill the space between culture chambers within a petri dish with culture medium.