This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.
I. Dissection of brains from adult fly
II. Mounting the CNS
III. Electrophysiology
The isolated whole brain preparation we illustrate in this video allows assessment of cellular mechanisms underlying excitability and synaptic transmission in identified neurons, including those in the olfactory processing pathways, in the adult Drosophila brain (Gu and O Dowd, 2006). This approach is complementary to study of neuronal activity in the brain of intact adult Drosophila (Wilson et all 2004), in much the same way recordings from neurons in a mammalian brain slice are complementary to recordings in awake behaving mammals. There are two major advantages of the in situ fly brain preparation when compared to the mammalian slices. First the entire fly brain fits easily into the recording chamber so it is not necessary to excise a small piece of tissue from a larger neural circuit that occurs when making mammalian brain slices. Therefore the neuronal circuits within the isolated Drospohila brain remain largely intact. Secondly, the cell bodies of most of the neurons are near the brain surface, readily accessible for whole cell recording and they remain functionally active when continuously perfused with oxygenated saline for several hours.
Using a glass bottom recording chamber also allows identification of specific neurons on the basis of their location and/or GFP expression. In this preparation, recording during perfusion requires stabilization of the brain. This is not compatible with standard procedures, including strategically placed gold wire or nylon mesh, that are effective for tissues such as hippocampal slices, due to the small size of the whole brain (~1mm). Therefore we a designed holder with a platinum frame and nylon cross hairs positioned to contact the brain in only two locations to minimize damage to neurons located near the brain surface. This stabilizes the brain, with either the anterior or posterior surface facing up and the opposite surface just lightly resting on the floor of the recording chamber. Using this device we are able to routinely maintain stable whole cell recordings for up to an hour, even from small neurons including Kenyon cells, while doing pharmacological manipulations that require bath application of specific drugs. The holder should also be useful in securing other small tissue samples for electrophysiological studies such as spinal cord slices from neonatal rodents.
This work was supported by an NIH grant NS27501 to DKOD. Additional support for this work was provided by an HHMI Professor Program Grant to DKOD.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
#5 Foceps | Tool | FST by DUMONT | No. 11251-10 | Be careful, never damage the fine tips of forceps |
Papain | Reagent | Worthington | 3126 | 20 U/ml activated by 1 mM L-cysteine |
Dissecting microscope | SMZ-2B Model:C-PS | Nikon | Equipped with gooseneck fiber optic lights positioned a low angle | |
Needle & Syringe | PrecisionGlide®?, Becton Dickinson & Co | 305175 & 309602 | Cheap and durable | |
Upright microscope | Axioskop2 FS plus | Zeiss | Equipped with a fixed stage, 40X water immersion objective, Nomarski optics, and fluorescent lamp | |
Patch clamp amplifier | 200 B | Molecular device | ||
Digidata D-A converter | 1322A | Molecular device |