In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.
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Method Article
Preparation of Drosophila Central Neurons for in situ Patch Clamping
In This Article
Summary
Abstract
Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.
Protocol
The following description is not specific for one motoneuron. It can be used with any neuron. In this example, we use the flight motoneuron 5 (MN5) that innervates the two dorsalmost fibers of the dorsolongitudinal wing depressor muscle (DLM). To identify and visualize MN5 we use the UAS/GAL4 system to express GFP in the flight motoneurons (and few other neurons).
1. Dissection of Adult Drosophila to Access the Dorsal Part of the Ventral Nerve Cord (VNC)
- Dissect an adult Drosophila dorsal side up along its dorsal midline in a sylgard coated Petri dish (35 mm diameter) as described in Ryglewski and Duch (2009
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Discussion
When visualizing the cells with fluorescent proteins like GFP, it is important to not over-expose the preparation to too much light. This may result in photo damage. We use 100W HBO short arc mercury bulbs for illumination, and we also use neutral density (ND) of 0.8 (Chroma ND filters 0.3 and 0.5). To be able to judge on the quality of the cleaning good visibility is crucial. Therefore, ND filters may be removed for short periods of about 20 s for multiple times.
When applying some positive .......
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Disclosures
No conflicts of interest declared.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Protease type XIV | Sigma Aldrich USA | P5147 | |
| Microfil flexible injection needle | World Precision Instruments USA | MF28G-5 | |
| Borosilicate Glass Capillaries, o.d. 1.5 mm, i.d. 1.0 mm, no filament | World Precision Instruments USA | PG52151-4 | |
| DiI | Invitrogen USA | D3899 | |
| Sylgard Elastomer Kit 184 (Dow Corning) | www.ellsworth.com | 184 SIL ELAST KIT | |
| ND filter set (unmounted) | Chroma Technology Corp. | 22000b series | |
| Electrode holder 1-HL-U | Molecular Devices | 1-HL-U |
References
- Papazian, D. M., Schwarz, T. L., Tempel, B. L., Jan, Y. N., Jan, L. Y. Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila. Science. 237, 749-753 (1987).
- Tempel, B. L., Papazian, D. M., Schwarz, T. L., Jan, Y. N., Jan, L. Y.
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