This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.
Preparations before day of culturing:
On day of culturing
I. Prepare Enzyme Solution (ES) in laminar flow hood
II. Make DDM2 media
On day of culturing: add the following supplements to make DDM2
To 10 ml of DMEM add the supplements, just before culturing:
Note: Make enough DDM2 for all cultures planned (each brain plated on a single coverslip in an individual 35 mm Petri dish, 1.5 ms of media/culture)
Step III-VI can be at a done nonsterile lab bench and should be completed in 45 minutes or less.
III. Pupae Collection
Note: We generally use brains from pupae between 55-78 hours after puparium formation (APF). It is slightly harder to dissect out the brain from the younger pupae since the head capsules tend to collapse, while the overall level of neurite outgrowth is slightly lower from the oldest pupae. In the Canton-S wildtype strain, these stages are recognized by pigmented eyes that are light brown to slightly reddish, with little pigment elsewhere.
IV. Decapitation under dissecting microscope
V. Removal of the brain from head under dissecting microscope
VI. Removal of optic lobes and enzyme treatment
Inside sterile laminar flow hood – all steps where tissue is exposed to air should be done in laminar flow hood for the remaining steps.
VII. Washing
VIII. Trituration and Plating under dissecting microscope
IX. Feeding Cells
Neurons harvested from the brains of embryonic/postnatal rodents can be grown in primary cell culture where they extend neurites and form functional synaptic connections. Methods for preparation of these cultures are well established and studies in rodent neuronal cultures have played a critical role in identifying genes and environmental factors involved in regulation of synapse formation and function (Banker and Goslin, 1991). While insect neurons from a variety of species can also be grown in culture, the only insect neurons shown to form functional synaptic connections in culture are from Drosophila (Rohrbough et al, 2003). Neuroblasts harvested mid-gastrula stage Drosophila embryos grown in defined media give rise to neurons that are electrically excitable and form functional, interneuronal synaptic connections (O’Dowd, 1995; Lee and O’Dowd, 1999). To identify factors that regulate synaptic form and function in neurons known to be involved in mediating adult behaviors we developed the technique illustrated in this video for harvesting and culturing neurons from brains of late stage Drosophila (Su and O’Dowd, 2003). One of the key features of this procedure was to use papain, instead of collagenase or other harsh enzymes traditionally used in preparation of insect neuronal cultures. Papain results in dissociated neurons retaining short axonal processes that survive, regenerate neuritic processes, and form functional interneuronal synaptic connections. Whole recordings in identified cell populations, including mushroom body Kenyon cells, show that fast excitatory synaptic transmission in the cultures is mediated by nAChRs while inhibition is mediated GABA receptors (Su and O’Dowd, 2003). Our recent electron microscopic analysis demonstrates that the neurons in culture form synapses with structural features that are similar to both chemical and electrical synapses in the adult brain (Oh et al., 2007). As illustrated in the video, the cultures are also amenable to Fura-2 calcium imaging studies and we have used these to investigate the cellular mechanisms underlying spontaneous and evoked changes in intracellular calcium levels in identified cell populations including Kenyon cells and cholinergic neurons (Jiang et al., 2005; Campusano et al., 2007). With the extensive genetic tool kit available in Drosophila this culture system provides a very useful tool for identifying intrinsic and extrinsic regulators of central synapse structure, function, and plasticity.
This work was supported by NIH grant NS27501 to DKOD. Additional support for this work was provided by a grant to UC Irvine in support of DKOD through the HHMI Professor Program.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Concanavalin A | Sigma | C-2010 | To 2.5 ml DS, add 25 mg Concanavalin A bottle. This is 10 mg/ml concentration. Make aliquots of 90 ul and store at -20 C, no longer than 3 months. | |
Laminin | Sigma | L-2020 | Add 1ml DS to 1mg Laminin bottle. This is 0.5 mg/ml concentration. Make aliquots of 10 ul and store at -20 C, no longer than 3 months. | |
Coverslips | Bellco Biological Glassware | 1943-00012 | 12 mm glass coverslips | |
ConA + Laminin solution | Stock solution, from Con A stock and laminin stock, for coverslip coating: Add in 5 ml DS, 83.5 ul of ConA (167 ug/ml) and 8.35 ul of Laminin (0.835 ug/ml). Mix. Make aliquots of 100 ul and store at -20 C, not longer than a month. | |||
Dissecting Solution | Buffer | For 500 ml: 400 ml Ultra filtered water + 25 ml Stock Solution A + 14 ml Stock Solution B + 3.0 g (33.3 mM) D (+)-Glucose (Sigma G-8270) + 7.5 g (43.8 mM) Sucrose (Sigma S-0389). Adjust pH to 7.4 with 1N NaOH (around 2 ml). Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Label “Dissecting Solution” and store at 4?C. | ||
Dissecting Solution | Buffer | For 500 ml: 400 ml Ultra filtered water + 25 ml Stock Solution A + 14 ml Stock Solution B + 3.0 g (33.3 mM) D (+)-Glucose (Sigma G-8270) + 7.5 g (43.8 mM) Sucrose (Sigma S-0389). Adjust pH to 7.4 with 1N NaOH (around 2 ml). Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Label “Dissecting Solution” and store at 4?C. | ||
Solution B: HEPES | Buffer | Sigma | H-3375 | 20.97g (9.9mM). Add ultra filtered water up to 200 ml. Mix until dissolve and bring final volume to 250 ml. Place in a clean bottle and autoclave. Label “Solution B” and store at 4 C. |
Solution A | Buffer | For 500 ml: 80.0g (137 mM) NaCl (Sigma S-9625) + 4.0g (5.4mM) KCl (Sigma P-4504) + 0.24g (0.17mM) Na2HPO4 (Sigma S-0876) + 0.3g (0.22 mM) KH2PO4 (Sigma P-5379). Weigh out all ingredients and mix until dissolved with 400 ml ultra filtered water. Bring final volume to 500 ml. Place in a clean bottle and autoclave. Label “Solution A” and store at 4°C. |