Name: Whole Cell Recordings from Brain of Adult Drosophila (Video) | JoVE Journal
Uploaded: 2007-07-29T00:00:00.000+00:00
Duration: 9 min 27 s
Description: Learn whole cell recording techniques for adult Drosophila brain neurons using GFP labeling. This method enables detailed electrophysiological analysis of single neurons in neurobiology research.

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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.

I. Dissection of brains from adult fly
<ol>
<li>Place a small drop (~100 &micro;l) of dissecting solution in center of a 35 mm Petri dish.</li>
<li>Catch adult female using aspirator. Under dissecting microscope, use two syringe needles to decapitate the fly.</li>
<li>Place head in a small drop of dissecting saline (with papain added) in a Petri dish.</li>
<li>Position head with with proboscis facing bottom of the dish.</li>
<li>Hold the cuticle covering the left compound eye with needle 1. Make a diagonal cut that extends from the dorsal to the ventral surface with needle 2, just medial to needle 1.</li>
<li>Hold the mouthparts with needle 1 and use needle 2 to make a horizontal cut that extends from the ventral surface of the left eye to the right eye, at the level of the base of proboscis.</li>
<li>Hold the cuticle covering the right compound eye with needle 1. Make a diagonal cut that extends from the dorsal to the ventral surface with needle 2, just medial of needle 1.</li>
<li>Rotate the head so that the rostral side is on the Petri dish surface. Insert needle 1 between the rostral cuticle and brain, and use needle 2 to follow the same path as the first needle. Ideally, the capsule will be peel away from the brain with both optic lobes attached since these are important in stabilizing the brain for recording.</li>
<li>The trachea, air sacs, and other connective tissues are carefully removed using two fine tip forceps.</li>
<li>Whole dissection should take 3-10 minutes</li>
</ol>
II. Mounting the CNS
<ol>
<li>The brain is transferred to the recording chamber using a yellow tip pipet.</li>
<li>In the recording chamber, the brain is stabilized by placing the platinum frame such that two fine cross hairs make contact with the tissue at the junction between the optic lobes and central brain region.</li>
<li>Each brain is allowed to rest in the recording chamber with continuous perfusion with oxygenated saline (95% oxygen and 5% carbon dioxide) for at least 10 minutes. Perfusion of the chamber with oxygenated saline is continued throughout the recording period. Preparation is visualized using an upright microscope( Axioskop 2FS; Zeiss, Oberkochen, Germany) with a fixed stage and a 40x water immersion objective (Achroplan; numerical aperture,0.8; Zeiss) and Nomarski optics. GFP was viewed with a BP 505-530 fluorescence filter.</li>
</ol>
III. Electrophysiology
<ol>
<li>Pipets of 8-14 Mohms are used for whole cell recordings</li>
<li>Current-clamp and voltage-clamp recordings are performed using a List EPC7 or an Axopatch 200B amplifier,a Digidata 1322A D-A converter (Molecular Devices, Foster City, CA), a Dell Dimension 8200 computer (Dell Computer, Round Rock, TX), and pClamp 9 software (Molecular Devices).</li>
</ol>

<ol><li>Gu, H., O Dowd, D. K., K, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cholinergic+synaptic+transmission+in+Adult+Drosophila+Kenyon+cells+in+situ%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Cholinergic synaptic transmission in Adult Drosophila Kenyon cells in situ.</a> Journal of Neuroscience. 26, 265-272 (2006).</li>
<li>Wilson, R. I., Turner, G. C., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transformation+of+olfactory+representations+in+the+Drosophila+antennal+lobe%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Transformation of olfactory representations in the Drosophila antennal lobe.</a> Science. 303, 366-370 (2004).</li>
</ol>

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 beha...

<table><tbody><tr><td>#5 Foceps</td><td>Tool</td><td>Fine Science Tools</td><td>No. 11251-10</td><td>Be careful, never damage the fine tips of forceps</td></tr><tr><td>Papain</td><td>Reagent</td><td>Worthington Biochemical</td><td>3126</td><td>20 U/ml activated by 1 mM L-cysteine</td></tr><tr><td>Dissecting microscope</td><td>SMZ-2B Model:C-PS</td><td>Nikon Instruments</td><td></td><td>Equipped with gooseneck fiber optic lights positioned a low angle</td></tr><tr><td>Needle &amp;amp; Syringe </td><td></td><td>PrecisionGlide&amp;#174;?, Becton Dickinson Co</td><td>305175 &amp;amp; 309602</td><td>Cheap and durable</td></tr><tr><td>Upright microscope</td><td>Axioskop2 FS plus</td><td>Carl Zeiss, Inc.</td><td></td><td>Equipped with a fixed stage, 40X water immersion objective, Nomarski optics, and fluorescent lamp</td></tr><tr><td>Patch clamp amplifier</td><td>200 B</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Digidata D-A converter </td><td>1322A</td><td>Molecular Devices</td><td></td><td></td></tr></tbody></table>

whole cell recordings from brain of adult drosophila

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

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.

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

My name is, I'm A assistant specialist in Department of Anatomy and Neurobiology in the uc R one. And today I will explain you the procedure, how to do the wholesale recording on the PHA brain, the key procedure of this preparation as how to de captive head, how to remove the brain and how to remove the TRICARE system and The G clear tissue from the surface of brain. Today I will introduce you how to make A dissection solution for the whole cell recording a adult soft lab brain.The first step is to make the, the second solution is really important, the key to make a good preparation for the adult brain. First I will put five mill of recorded solution in this cent field tube. And then here I have another small tube.I will add one mill of recorded solution in this tube there, have some sing and then I will make this sing solution. I will pipe it up and down to make a good sing solution. And then I will take 100 unit of of pepane from this bottle.And then I will put this pepane into this Centro field tube. And then you can see here the solution is cloudy and then I will put another 100 macro liter of cy and then I just mix that thoroughly. 10 minutes later the solution will be clear a

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Whole Cell Recordings from Brain of Adult Drosophila

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