21 Insecta
245
ment reminiscent of a synapse characterized the ending of an axon on
another axon. In addition to axons, glia, tracheoblasts and neurons
showed outgrowth. Explants developed rich cellular and axonal interconnections (Fig. 3).
Subprotocol 5
Nervous System of Adult Cockroaches (Blattaria)
Materials
- Dissecting tray (see chapter 1, section 3 "Tools")
- Scissors
- Forceps
- Insect pins
- Petri dishes
- Incubator
- Pasteur pipettes
Equipment
- Culture medium (a mixture of 5 parts Schneider's Drosophila medium Solutions
(see Appendix 1) and 4 parts minimal essential medium (MEM) with
Hanks' salts, supplemented with 25 mmolll HEPES, 1 % glutamine,
100 IV/ml penicillin, 100 Jlg/ml streptomycin, 0.25 glml fungizone,and
0.8% methyl cellulose)
- Collagenase (type IS; Sigma)
- Adults of P. americana
Animals
Procedure
1. Anaesthetize cockroaches by holding them under water for 4 min.
Sterilization
2. Surface sterilize them by immersion in 70% ethanol for 10 min.
1. Fix the cockroach, ventral side uppermost, onto a dissecting tray us- Dissection
ing several insect pins.
2. Remove the legs and the cuticle of the thorax to expose the meso- and
metathoracic ganglia.
3. Excise these ganglia, and free them of adhering fat bodies and tracheal tissues.
4. Rinse the ganglia in culture medium with 5x concentrated antibiotics
and without methyl cellulose.
1. Transfer the ganglia into a dish containing 0.5 ml of 0.5%-1.0% colla- Digestion
genase, and incubate at 29°C for 1.5 h. The concentration of the collagenase should be predetermined by combination of the use of serially
diluted enzyme and the varied incubation period.
245
ment reminiscent of a synapse characterized the ending of an axon on
another axon. In addition to axons, glia, tracheoblasts and neurons
showed outgrowth. Explants developed rich cellular and axonal interconnections (Fig. 3).
Subprotocol 5
Nervous System of Adult Cockroaches (Blattaria)
Materials
- Dissecting tray (see chapter 1, section 3 "Tools")
- Scissors
- Forceps
- Insect pins
- Petri dishes
- Incubator
- Pasteur pipettes
Equipment
- Culture medium (a mixture of 5 parts Schneider's Drosophila medium Solutions
(see Appendix 1) and 4 parts minimal essential medium (MEM) with
Hanks' salts, supplemented with 25 mmolll HEPES, 1 % glutamine,
100 IV/ml penicillin, 100 Jlg/ml streptomycin, 0.25 glml fungizone,and
0.8% methyl cellulose)
- Collagenase (type IS; Sigma)
- Adults of P. americana
Animals
Procedure
1. Anaesthetize cockroaches by holding them under water for 4 min.
Sterilization
2. Surface sterilize them by immersion in 70% ethanol for 10 min.
1. Fix the cockroach, ventral side uppermost, onto a dissecting tray us- Dissection
ing several insect pins.
2. Remove the legs and the cuticle of the thorax to expose the meso- and
metathoracic ganglia.
3. Excise these ganglia, and free them of adhering fat bodies and tracheal tissues.
4. Rinse the ganglia in culture medium with 5x concentrated antibiotics
and without methyl cellulose.
1. Transfer the ganglia into a dish containing 0.5 ml of 0.5%-1.0% colla- Digestion
genase, and incubate at 29°C for 1.5 h. The concentration of the collagenase should be predetermined by combination of the use of serially
diluted enzyme and the varied incubation period.