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 stimulating electrode micromanipulators as shown in Figure 2. </li>
 <li> Before starting the experiment, ensure that you have a comfortable range on all axes of rotation with all micromanipulators, and that all electrodes as well as the injection micropipette (see step 2) can reach the animal. </li>
</ol>
2. Nanoinjection Set-up
<ol>
 <li>The nanoinjection set-up requires a Nanoliter2000 (World Precision Instruments, Sarasota, FL, U.S.A.) or similar type of injector that allows controlled injections in nanoliter quantities. </li>
 <li> Prepare injection needle using the glass micropipettes supplied with the injector by pulling them to a resistance of 80-100 M&Omega; with an electrode puller. </li>
 <li> For smooth injections it is required to bevel the micropipettes to an 11-17 &mu;m opening at a 45 degree angle (Figure 3). </li>
 <li> Slowly backfill the injection micropipette with synthetic oil using a Hamilton syringe as instructed by the Nanoliter2000 manual, ensuring that no air bubbles are present. </li>
 <li> Carefully secure the micropipette on the nanoinjector and prepare it to load the compound by emptying excess oil as instructed by the Nanoliter2000 manual. </li>
 <li> Place the injector on the micromanipulator and load the compound as instructed in the Nanoliter2000 manual. Make sure that the tip of the micropipette does not break during this procedure. </li>
 <li> Set the desired amount of nanoliters to be injected in the injector's control box as instructed by the Nanoliter2000 manual. Please note that the total amount injected should not exceed 100 nl. We found that larger quantities of saline control solutions may affect the function of the GFS circuit. </li>
 <li> It is critical to have the injector unplugged from the control box during recording acquisition with the exception of the injection itself, since the power supply of the nanoinjector interferes with the recordings, which is visible as background noise (Figure 4). However, do not disconnect the power supply to the control box as it will re-set it. </li>
</ol>
3. Drosophila melanogaster Preparation
<ol>
 <li> Anesthetize 2 to 6 day old flies with CO2 or on ice as previously described14,15. </li>
 <li> Once immobile, use a pair of tweezers to transfer the animal to a small plate with soft dental wax by picking it up from its legs. Please note that a male fruit fly weighs about 1.0 mg and a female fruit fly weighs about 1.2 mg, thus compound-to-body weight ratio is different in male versus female flies. Therefore, it is recommended to use only one gender for the experiments. </li>
 <li>As previously described14,15, carefully mount the fly dorsal side up and ensure that the thorax and head are immobilized with soft dental wax placed around the body. Carefully spread the wings out so that they lay perpendicular to the thorax (Figure 2, C). The fly should be mounted with as little damage as possible. </li>
</ol>
4. Paired Nanoinjection/electrophysiology 
<ol>
 <li> Place the mounted fly on the electrophysiology rig with its head towards the stimulating electrodes.</li>
 <li> Impale the animal with the corresponding stimulating, ground and recording electrodes as previously described14,15 (Figure 2, C). Unless otherwise desired, place one recording electrode into the DLM and the other into the TTM muscle. The DLM muscle is located in the thorax between the anterior Dorso-Central hairs and the midline of the fly. The TTM muscle is located near the wing attachments, between the posterior and anterior Supra-Alar hairs of the fly7.</li>
 <li> Align the injection micropipette containing the compound with the center of the three ocelli situated on the medial posterior portion of the head but do not inject yet (Figure 2, C). </li>
 <li> Before compound injection obtain a baseline recording of the GF to TTM and GF to DLM pathways of the Giant Fiber System (GFS, Figure 1) via brain stimulation. To do so, activate the Giant Fibers (GFs) with 10 trains of 10 stimuli (40-50 mV) each given for a duration of 0.03 ms at 100 Hz with a 1 second delay between the trains14,15 (Figure 4). Wild type flies should be able to follow one-to-one at this rate of stimulation for both DLM and TTM pathways. Discard the fly if the GF to DLM and GF to TTM pathways do not follow the 100 Hz stimulation at a 1:1 ratio. </li>
 <li> Switch to continuous stimulation of the GF with single pulses at 1 Hz (Figure 4). </li>
 <li> Quickly plug the injector into the control box. Even though background noise will interfere with the 1 Hz stimulation recordings, do not discontinue it. </li>
 <li> Carefully insert the injection micropipette into the head capsule of the fly just below the cuticule, and inject the desired amount of compound into the hemolymph of the fly while maintaining the 1 Hz stimulation (Figure 4). Due to the open circulatory system of the fly, the entire nervous system will be exposed to the compound within seconds. Though a particular injection site is not critical to deliver compounds into the hemolymph, we find the region of the ocelli, which are localized medial at the most dorsal side of the head capsule, to be a convenient site that allows for an easy injection that leads to rapid and even distribution of the compound. </li>
 <li> Immediately remove injection micropipette from site of injection and unplug the injector from the control box while continuing the 1 Hz GF stimulation up to 1 min after injection (Figure 4). </li>
 <li> In order to reveal more subtle effects of compounds on the GFS, stress the Giant Fibers (GFs) with 10 trains of 10 stimuli at 100 Hz with a 1 second delay between the trains. Continue to test the function of the GF pathways with this paradigm every 5 minutes up to 15 minutes (Figure 4). However, shorter intervals or longer monitoring periods are also possible. </li>
 <li> In order to test whether the compound has an effect at the neuromuscular junctions (NMJs) of the GFS, and possibly narrow down the effects of the compound, proceed to activate the motor neurons directly by thoracic stimulation. For this, remove the stimulating electrodes from the eyes and replace them on the anterior sides of the thorax in order to stimulate the motor neurons with 10 trains of 10 stimuli at 100 Hz. </li>
</ol>
Note: The electrophysiology traces shown in the video do not correspond to the effects of pure dye injection. 
5. Representative Results
Effect of an antagonist on the PSI to DLM synapse of the Giant Fiber System
Methyllycaconitine citrate (MLA) is a nAChR antagonist that is specific for &alpha;7 nAChR subtypes. The PSI to DLMn synapse in the GF-DLM pathway is dependent on the D&alpha;7 nAChR subtype for proper function, while genetic removal of D&alpha;7 nAChR subtype has no effect on the GF-TTM pathway5,6. In order to demonstrate the specificity and sensitivity of our assay we injected MLA at different concentrations (0, 0.02, 0.04, 0.08, 0.12 ng/mg, 46 nl injected) into the head of the animal (n=10 per compound treatment; n=15 for saline treatment). Only male flies (of the wild type genotype wild 10E) were used, and the effect of the compound was monitored for a total of 15 minutes after injection. 
Figure 5 depicts the difference between baseline recordings obtained before injection and those obtained after injection in response to MLA and saline control solution. We found that injection of MLA resulted in the inability of the GF-DLM pathway to follow one-to-one at 100 Hz by stimulations of the GFs in the brain while the GF-TTM pathway remained unaffected. (Figure 5, Top and middle trace, t-test performed between saline controls [0 ng/mg] and the different concentrations of MLA at each time point unless the data is non-parametric [normality and equal variances tested], otherwise we use a Mann-Whitney Rank Sum Test. *p&lt;0.001). However, a one-to-one response of the DLM was observed when the motor neurons were stimulated directly (Figure 5, bottom trace), demonstrating that the NMJ function of the DLM and TTM is not affected by MLA. MLA appeared to reach its maximum effect 1 minute after injection for 0.04, 0.08 and 0.12 ng/mg of MLA injected, as no further significant changes were noted during the following 15 minutes of testing period. Moreover, the compound reached a maximum effect at 0.08 ng/mg since stronger responses were not observed with the higher dosage of 0.12 ng/mg.
<img src="/files/ftp_upload/3597/3597fig1.jpg" alt="Figure 1" /> 
Figure 1. The Giant Fiber System Diagram of the Giant Fiber System (GFS). The Giant Fibers (GFs, shown in red) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI, shown in green) and the Tergo Trochanteral Muscle neuron (TTMn, shown in yellow)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron, shown in blue) connection is dependent on D&alpha;7 nAChR subtype6. Finally the neuromuscular junction (NMJ) of the TTMn and the DLMn onto the jump (TTM, shown in purple) and flight muscles (DLM, shown in purple) is glutamatergic.
Note: The GF to PSI connection is both electrical and chemical. However, in shakB mutants (which lack gap junctions), no response can be recorded from the DLM upon stimulation of the GFs in the brain, demonstrating that the chemical component in the absence of electrical connections is not sufficient to evoke an action potential in the PSI 5,16-18. Because the GF to PSI connection is gap junction dependent, this figure shows only the GAP junction at the synapse for simplicity reasons. 
<img src="/files/ftp_upload/3597/3597fig2.jpg" alt="Figure 2" /> 
Figure 2. 
 Micromanipulators set-up. 
<ol class="ualpha">
<li>A modified set-up of a previously published protocol14 is used to fit the injection micromanipulator for paired GFS recordings with simultaneous nanoinjections. The mounted fly preparation is oriented with the head of the fly towards the experimenter. The injection micromanipulator (#1) is placed in front of the experimenter between the two manipulators for the tungsten stimulating electrodes (2# and #3). The two micromanipulators for the glass recording electrodes (#4 and #5) are placed on the left and right side, respectively. The micromanipulator for the tungsten ground electrode (#6) is placed furthest in the back either on the left side (shown here) or on the right side.</li>
<li> A close up view from the top of the arrangements of the various electrodes and injection micropipette.</li>
<li>A properly mounted D. melanogaster impaled with electrodes and injection micropipette ready for injection. Notice that the animal's body is mounted with its thorax horizontally and its wings spread out. The wax is securely wrapped around its body preventing the animal from moving. Additionally, the ground electrode (#6, in the abdomen), the glass recording electrodes (#4 and #5, in the thorax, highlighted by dark outlines), and the stimulating electrodes (#2 and #3, one in each eye) are impaled in place, as previously described14. The injection micropipette (#1) is properly aligned with the center of the three ocelli (circled). Insertion of the injection micropipette should be placed in this area.</li>
</ol>
<img src="/files/ftp_upload/3597/3597fig3.jpg" alt="Figure 3" /> 
 Figure 3. Beveled injection micropipette. A diagram of a properly beveled micropipette is shown here. The electrode opening should be beveled at a 45 degree angle and have an opening between 11 to 17 &mu;m. A proper beveled injection micropipette is crucial for a smooth injection with minimal damage to the fly.
<img src="/files/ftp_upload/3597/3597fig4.jpg" alt="Figure 4" /> 
Figure 4. Overall scheme of the nanoinjection/electrophysiology protocol. A representative diagram of the overall scheme for the nanoinjection/electrophysiology protocol. Start by obtaining a baseline recording by stimulating the Giant Fibers (GFs) at 100 Hz with 10 trains of 10 stimuli each (only one train shown here). Before injection, begin the 1 Hz stimulations one second apart. During injection time (while injector is plugged in to the control box), you will observe significant background noise; however, do not discontinue the recordings. After injection (and injector is unplugged from control box), continue the 1 Hz stimulation for about 1 more minute. Finally, proceed to stress the GFs with 10 trains of 10 stimuli at 100 Hz and continue to test the function of the GF pathways with this paradigm every 5 minutes up to 15 minutes. Note: recordings were manipulated to create the overall scheme and do not represent a specific result obtained. Not to scale, not all traces are shown. <a href="https://www.jove.com/files/ftp_upload/3597/3597fig4large.jpg">Click here for larger image</a>.
<img src="/files/ftp_upload/3597/3597fig5.jpg" alt="Figure 5" /> 
Figure 5. The effects of MLA in the GFS. 
<ol class="ualpha">
 <li> A graphical depiction of the effects of the &alpha;7 nAChR antagonist Methyllycaconitine citrate (MLA) on the GF-DLM pathway of the fruit fly at different concentrations (0, 0.02, 0.04, 0.08, 0.12 ng/mg. n=10 per compound treatment; n=15 for saline treatment). Only one minute after injection, a significant and immediate effect was seen with 0.04 ng/mg of MLA. A significant effect was also seen with 0.8ng/mg and 0.12ng/mg of MLA at 100Hz stimulation of the GFs. No significant difference was noted between saline controls and 0.02 ng/mg of MLA. Additionally, no change in effect was seen after 1 minute post injection during the time tested (15 minutes). A t-test was performed between saline controls (0 ng/mg) and the different concentrations of MLA at each time point. Levels are reported as mean +/- SEM, *p &lt; 0.001. </li>
 <li> Sample traces of the DLM responses at 100 Hz stimulation. Top trace shows the responses of the muscle before MLA injection upon GF stimulation in the brain. Notice that the muscle is able to respond to each stimulus one-to-one at 100 Hz. Middle trace displays the responses of the DLM after MLA injection (0.12 ng/mg). Notice that the muscle is not able to respond to each stimulus one-to-one at 100 Hz. (asterisks). Bottom trace shows the responses of the DLM of the same preparation (0.12 ng/mg) upon direct stimulation of the motor neurons in the thorax. Because DLM responds one-to-one at 100 Hz with thoracic stimulation, the failure of responses with brain stimulations can be attributed to the cholinergic PSI-DLMn connection. </li>
 <li> A graphical depiction of the effects with different MLA concentrations (0, 0.02, 0.04, 0.08, 0.12 ng/mg) on the GF-TTM pathway. No significant effects were seen between saline (0 ng/mg) and compound injections at any time point. A t-test was performed between saline controls (0 ng/mg) and the different concentrations of MLA at each time point, *p &lt; 0.001. </li>
 <li> Sample traces of the TTM responses at 100 Hz stimulation. Top trace shows the responses of the muscle before MLA injection upon GF activation with stimulation in the brain. Notice that the muscle is able to respond to all stimuli at 100 Hz. Middle trace shows the responses of the DLM after MLA injection (0.12 ng/mg). Responses from the TTM muscle to stimulation of the GF in the brain remain one-to-one. Bottom trace shows the responses of the TTM of the same preparation to 100 Hz stimulation of the motor neurons in the thorax (0.12 ng/mg). </li>
</ol>

<ol>
	<li>Koehn, F. E., Carter, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+role+of+natural+products+in+drug+discovery.">The evolving role of natural products in drug discovery.</a> Nat. Rev. Drug Discov. 4, 206-220 (2005).</li><li>Miljanich, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ziconotide:+neuronal+calcium+channel+blocker+for+treating+severe+chronic+pain.">Ziconotide: neuronal calcium channel blocker for treating severe chronic pain.</a> Curr. Med. Chem. 11, 3029-3040 (2004).</li><li>Layer, R. T., Wagstaff, J. D., White, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conantokins:+peptide+antagonists+of+NMDA+receptors.">Conantokins: peptide antagonists of NMDA receptors.</a> Curr. Med. Chem. 11, 3073-3084 (2004).</li><li>Lewis, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conotoxins+as+selective+inhibitors+of+neuronal+ion+channels,+receptors+and+transporters.">Conotoxins as selective inhibitors of neuronal ion channels, receptors and transporters.</a> IUBMB Life. 56, 89-93 (2004).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, M. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Fayyazuddin, A., Zaheer, M. A., Hiesinger, P. R., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nicotinic+acetylcholine+receptor+Dalpha7+is+required+for+an+escape+behavior+in+Drosophila.">The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila.</a> PLoS biology. 4, e63 (2006).</li><li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-glutamate+as+an+excitatory+transmitter+at+the+Drosophila+larval+neuromuscular+junction.">L-glutamate as an excitatory transmitter at the Drosophila larval neuromuscular junction.</a> The Journal of physiology. 262, 215-236 (1976).</li><li>Usherwood, P. N., Machili, P., Leaf, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-Glutamate+at+insect+excitatory+nerve-muscle+synapses.">L-Glutamate at insect excitatory nerve-muscle synapses.</a> Nature. 219, 1169-1172 (1968).</li><li>Marrus, S. B., Portman, S. L., Allen, M. J., Moffat, K. G., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+localization+of+glutamate+receptor+subunits+at+the+Drosophila+neuromuscular+junction.">Differential localization of glutamate receptor subunits at the Drosophila neuromuscular junction.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 24, 1406-1415 (2004).</li><li>Petersen, S. A., Fetter, R. D., Noordermeer, J. N., Goodman, C. S., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+glutamate+receptors+in+Drosophila+reveals+a+retrograde+signal+regulating+presynaptic+transmitter+release.">Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release.</a> Neuron. 19, 1237-1248 (1997).</li><li>Qin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+different+subunits+are+essential+for+expressing+the+synaptic+glutamate+receptor+at+neuromuscular+junctions+of+Drosophila.">Four different subunits are essential for expressing the synaptic glutamate receptor at neuromuscular junctions of Drosophila.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 25, 3209-3218 (2005).</li><li>Schuster, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+an+invertebrate+glutamate+receptor+subunit+expressed+in+Drosophila+muscle.">Molecular cloning of an invertebrate glutamate receptor subunit expressed in Drosophila muscle.</a> Science. 254, 112-114 (1991).</li><li>Tanouye, M. A., Wyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+outputs+of+giant+nerve+fiber+in+Drosophila.">Motor outputs of giant nerve fiber in Drosophila.</a> Journal of. 44, 405-421 (1980).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. (47), e2412 (2011).</li><li>Allen, M. J., Godenschwege, T., Zhang, B., Freeman, M. R., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Neurobiology. , 215-224 (2010).</li><li>Blagburn, J. M., Alexopoulos, H., Davies, J. A., Bacon, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Null+mutation+in+shaking-B+eliminates+electrical,+but+not+chemical,+synapses+in+the+Drosophila+giant+fiber+system:+a+structural+study.">Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: a structural study.</a> J. Comp. Neurol. 404, 449-458 (1999).</li><li>Thomas, J. B., Wyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+altering+synaptic+connectivity+between+identified+neurons+in+Drosophila.">Mutations altering synaptic connectivity between identified neurons in Drosophila.</a> J. Neurosci. 4, 530-538 (1984).</li><li>Baird, D. H., Schalet, A. P., Wyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Passover+locus+in+Drosophila+melanogaster:+complex+complementation+and+different+effects+on+the+giant+fiber+neural+pathway.">The Passover locus in Drosophila melanogaster: complex complementation and different effects on the giant fiber neural pathway.</a> Genetics. 126, 1045-1059 (1990).</li><li>Gorczyca, M., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+cholinergic+synapse+in+the+giant+fiber+pathway+of+Drosophila+using+conditional+mutations+of+acetylcholine+synthesis.">Identification of a cholinergic synapse in the giant fiber pathway of Drosophila using conditional mutations of acetylcholine synthesis.</a> J. Neurogenet. 1, 289-313 (1984).</li><li>Allen, M. J., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+component+of+the+mixed+GF-TTMn+synapse+in+Drosophila+melanogaster+uses+acetylcholine+as+its+neurotransmitter.">The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter.</a> The European journal of neuroscience. 26, 439-445 (2007).</li><li>Mejia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+for+in+vivo+screening+of+toxins+using+the+Drosophila+Giant+Fiber+circuit.">A novel approach for in vivo screening of toxins using the Drosophila Giant Fiber circuit.</a> Toxicon. 56, 1398-1407 (2010).</li><li>Stork, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+function+of+the+blood-brain+barrier+in+Drosophila.">Organization and function of the blood-brain barrier in Drosophila.</a> J. Neurosci. 28, 587-597 (2008).</li></ol>

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

13.1K Views.  Florida Atlantic University. Un rapido In vivo Test per verificare i composti neuromodulatori che utilizzano il sistema di Giant Fiber (GFS) di Drosophila melanogaster Descritto. Nanoinjections nella testa dell'animale insieme registrazioni elettrofisiologiche di GFS può rivelare la bioattività dei composti sui neuroni e muscoli.

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

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...