We would like to acknowledge the members of the Mari lab and the Godenschwege lab, in particular Aline Yonezawa, for comments and help with this protocol. This work was funded by the National Institute for Neurological Disorders and Stroke grant R21NS06637 to F.M. and T.A.G.; A.B. was funded by the National Science Foundation award number 082925, URM: Integrative Biology for future researchers.

1. Electrophysiology Rig Set-up
<ol>
 <li> The required equipment for the electrophysiology rig set-up is described in detail by Augustin et al. in this Journal14. Please refer to this article for a detailed explanation of the electrophysiological apparatuses needed. </li>
 <li> Modify the previously described electrophysiology rig set-up14 by adding a sixth micromanipulator, which holds the nanoinjector. For easy access to the head of the animal it should be placed between the two s...

<ol>
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No conflicts of interest declared.

The nanoinjection/electrophysiology bioassay presented here allows for a rapid screening of compounds in the nervous system of the fruit fly. This is a novel in vivo technique that requires small quantities of a compound to elicit an effect on a variety of molecular targets in a well-characterized neuronal circuit. This method can be used to test the bioactivity of different compounds, from unknown toxins to commercially available pharmacological agents. 
Here we demonstrated the func...

<table border="1">
<tbody>
 <tr>
 <td>Name 			of the reagent</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Recording 			glass electrodes: borosilicate 			glass capillaries</td>
 <td>World 			Precision Instruments</td>
 <td>1B100F-4</td>
 <td>1.0mm OD, 			0.58mm ID</td>
 </tr>
 <tr>
 <td>Stimulator </td>
 <td>Grass 			Instruments</td>
 <td>Model S48</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Getting 			Instruments Inc.</td>
 <td>Model 5A</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Data 			acquisition Software: Digidata</td>
 <td>Molecular 			Devices</td>
 <td>Model 1440A</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Data 			collection software: pCLAMP</td>
 <td>Molecular 			Devices</td>
 <td>Version 10</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Stereomicroscope 			with fiber optic microscope ring illuminator </td>
 <td>AmScope </td>
 <td>SM-4T 
Model 			HL250-AR </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Dissecting 			scope for mounting</td>
 <td>AmScope</td>
 <td>SM-2TZ</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Kite Manual 			Micromanipulator &amp; Tilting Base </td>
 <td>World 			Precision Instruments</td>
 <td>Model 			# M3301 
Kite: 			Model # KITE-M3-L </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Drosophila 			melanogaster Wild 10E genotype (wild type strain)</td>
 <td>Bloomington 			Stock center</td>
 <td>Stock # 3892 </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Vertical 			pipette puller</td>
 <td>David Kopf 			Instruments</td>
 <td>Model 700c</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Injection 			glass micropipettes: Borosilicate glass capillaries</td>
 <td>World 			Precision Instruments</td>
 <td>Catalogue # 			4878</td>
 <td>1.14mm OD, 			0.5mm ID</td>
 </tr>
 <tr>
 <td>Silicon 			oil </td>
 <td>Fisher</td>
 <td>Catalogue # 			S159-500 </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Beveler</td>
 <td>Sutter 			Instrument Co.</td>
 <td>K.T. 			Brown Type 
Model # BV-10</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Nanoliter2000</td>
 <td>World 			Precision Instruments</td>
 <td>Catalogue # 			B203XVY</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Blue food 			coloring</td>
 <td>McCormick</td>
 <td>N/A </td>
 <td>Ingredients: 			Water, Propylene Glycol, FD&amp;C Blue 1, and 0.1% Propylparaben 			(preservative).</td>
 </tr>
 <tr>
 <td>Methyllycaconitine 			citrate (MLA)</td>
 <td>Tocris 			Bioscience</td>
 <td>Catalogue # 			1029</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td> Plastic wax 			sticks</td>
 <td> Hygenic Corporation (Akron Ohio USA)</td>
 <td>&nbsp; </td>
 <td>&nbsp; </td>
 </tr>
 </tbody>
</table>

paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the drosophila melanogaster giant fiber system

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Watch this Scientific Journal Video about Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System at JoVE.com

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

paired-nanoinjection-electrophysiology-assay-to-screen-for

Research

JoVE Journal

Neuroscience

13.0K 조회수.  Florida Atlantic University. 급속한 생체내에서 검정의 거대한 섬유 시스템 (GFS)를 사용 neuromodulatory 화합물을위한 테스트 Drosophila melanogaster이 설명되어 있습니다. GFS의 electrophysiological 기록과 함께 동물의 머리에 Nanoinjections는 신경이나 근육에있는 화합물의 bioactivity를 공개하실 수 있습니다.