We would like to thank Ms. Rui Chen for the dissection and images of the Drosophila CNS shown in the figures.

1. Equipment and Materials
<ol>
	<li>Prepare the required components (listed in Table of Materials) of the electrophysiology rig to perform suction electrode recordings of the Drosophila CNS. 
	NOTE: Prior to experimentation, it is necessary to construct chambers for dissection of the Drosophila CNS and to be used for bathing the ganglia in saline during recordings. A step-by-step outline of chamber construction is provided below.</li>
	<li>Prepare the larval chamber.
	<ol>
		<li...

<ol>
	<li>Sparks, T. C., Nauen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRAC:+Mode+of+action+classification+and+insecticide+resistance+management.">IRAC: Mode of action classification and insecticide resistance management.</a> Pesticide Biochemistry and Physiology. 121, 122-128 (2015).</li><li>Swale, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insecticide+resistance-breaking+mosquitocide+targeting+inward+rectifier+potassium+channels+in+vectors+of+Zika+virus+and+malaria.">An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria.</a> Scientific Reports. 6, 36954 (2016).</li><li>Troczka, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+expression+and+functional+characterisation+of+the+diamondback+moth+ryanodine+receptor+G4946E+variant+conferring+resistance+to+diamide+insecticides.">Stable expression and functional characterisation of the diamondback moth ryanodine receptor G4946E variant conferring resistance to diamide insecticides.</a> Scientific Reports. 5, 14680 (2015).</li><li>Bloomquist, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+potassium+KV2+channels+as+new+targets+for+insecticides.">Voltage-sensitive potassium KV2 channels as new targets for insecticides.</a> Biopesticides: State of the Art and Future Opportunities. 1172, 71-81 (2014).</li><li>Jenson, L. J., Bloomquist, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+serum+and+ion+channel+block+on+growth+and+hormonally-induced+differentiation+of+Spodoptera+frugiperda+(Sf21)+insect+cells.">Role of serum and ion channel block on growth and hormonally-induced differentiation of Spodoptera frugiperda (Sf21) insect cells.</a> Archives of Insect Biochemistry and Physiology. 90 (3), 131-139 (2015).</li><li>Jenson, L. J., Sun, B., Bloomquist, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+potassium+channels+expressed+after+20-Hydroxyecdysone+treatment+of+a+mosquito+cell+line.">Voltage-sensitive potassium channels expressed after 20-Hydroxyecdysone treatment of a mosquito cell line.</a> Insect Biochemistry and Molecular Biolology. 87, 75-80 (2017).</li><li>Chintapalli, V. R., Wang, J., Dow, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+FlyAtlas+to+identify+better+Drosophila+melanogaster+models+of+human+disease.">Using FlyAtlas to identify better Drosophila melanogaster models of human disease.</a> Nature Genetics. 39 (6), 715-720 (2007).</li><li>Duffy, J. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larvalign:+Aligning+gene+expression+patterns+from+the+larval+brain+of+Drosophila+melanogaster.">Larvalign: Aligning gene expression patterns from the larval brain of Drosophila melanogaster.</a> Neuroinformatics. 16 (1), 65-80 (2017).</li><li>Sprecher, S. G., Reichert, H., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+patterns+in+primary+neuronal+clusters+of+the+Drosophila+embryonic+brain.">Gene expression patterns in primary neuronal clusters of the Drosophila embryonic brain.</a> Gene Expression Patterns. 7 (5), 584-595 (2007).</li><li>Zhou, S., et al. <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+model+for+toxicogenomics:+Genetic+variation+in+susceptibility+to+heavy+metal+exposure.">A Drosophila model for toxicogenomics: Genetic variation in susceptibility to heavy metal exposure.</a> PLoS Genetics. 13 (7), e1006907 (2017).</li><li>Manev, H., Dimitrijevic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+model+for+in+vivo+pharmacological+analgesia+research.">Drosophila model for in vivo pharmacological analgesia research.</a> European Journal of Pharmacology. 491 (2-3), 207-208 (2004).</li><li>Pandey, U. B., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+disease+models+in+Drosophila+melanogaster+and+the+role+of+the+fly+in+therapeutic+drug+discovery.">Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery.</a> Pharmacological Reviews. 63 (2), 411-436 (2011).</li><li>Hekmat-Scafe, D. S., Lundy, M. Y., Ranga, R., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+K+/Cl-+cotransporter+gene+kazachoc+(kcc)+increase+seizure+susceptibility+in+Drosophila.">Mutations in the K+/Cl- cotransporter gene kazachoc (kcc) increase seizure susceptibility in Drosophila.</a> Journal of Neuroscience. 26 (35), 8943-8954 (2006).</li><li>Whitworth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glutathione+S-transferase+activity+rescues+dopaminergic+neuron+loss+in+a+Drosophila+model+of+Parkinson's+disease.">Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease.</a> Proceedings of the National Academy of Science of the United States of America. 102 (22), 8024-8029 (2005).</li><li>Watson, M. R., Lagow, R. D., Xu, K., Zhang, B., Bonini, N. 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+Drosophila+model+for+amyotrophic+lateral+sclerosis+reveals+motor+neuron+damage+by+human+SOD1.">A Drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1.</a> Journal of Biological Chemistry. 283 (36), 24972-24981 (2008).</li><li>Rajendra, T. K., et al. <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+melanogaster+model+of+spinal+muscular+atrophy+reveals+a+function+for+SMN+in+striated+muscle.">A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle.</a> Journal of Cell Biology. 176 (6), 831-841 (2007).</li><li>Chan, H. Y., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+models+of+human+neurodegenerative+disease.">Drosophila models of human neurodegenerative disease.</a> Cell Death and Differentiation. 7 (11), 1075-1080 (2000).</li><li>Limmer, S., Weiler, A., Volkenhoff, A., Babatz, F., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+blood-brain+barrier:+development+and+function+of+a+glial+endothelium.">The Drosophila blood-brain barrier: development and function of a glial endothelium.</a> Frontiers in Neuroscience. 8, 365 (2014).</li><li>Rickert, C., Kunz, T., Harris, K. L., Whitington, P. M., Technau, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+characterization+of+the+entire+interneuron+population+reveals+principles+of+neuromere+organization+in+the+ventral+nerve+cord+of+Drosophila.">Morphological characterization of the entire interneuron population reveals principles of neuromere organization in the ventral nerve cord of Drosophila.</a> Journal of Neuroscience. 31 (44), 15870-15883 (2011).</li><li>Schwabe, T., Li, X., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+the+mesenchymal-epithelial+transition+of+blood-brain+barrier+forming+glia+in+Drosophila.">Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila.</a> Biology Open. 6 (2), 232-243 (2017).</li><li>Abbott, N. J., Ronnback, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-endothelial+interactions+at+the+blood-brain+barrier.">Astrocyte-endothelial interactions at the blood-brain barrier.</a> Nature Reviews Neuroscience. 7 (1), 41-53 (2006).</li><li>Abbott, N. J., Dolman, D. E., Patabendige, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+to+predict+drug+permeation+across+the+blood-brain+barrier,+and+distribution+to+brain.">Assays to predict drug permeation across the blood-brain barrier, and distribution to brain.</a> Current Drug Metabolism. 9 (9), 901-910 (2008).</li><li>Swale, D. R., Sun, B., Tong, F., Bloomquist, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxicity+and+mode+of+action+of+N,+N-diethyl-meta-toluamide+(DEET).">Neurotoxicity and mode of action of N, N-diethyl-meta-toluamide (DEET).</a> PLoS One. 9 (8), e103713 (2014).</li><li>Hafer, N., Schedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+larval+CNS+in+Drosophila+melanogaster.">Dissection of larval CNS in Drosophila melanogaster.</a> Journal of Visualized Experiments. (1), 85 (2006).</li><li>Bloomquist, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+of+atracotoxin+at+central+and+peripheral+synapses+of+insects.">Mode of action of atracotoxin at central and peripheral synapses of insects.</a> Invertebrate Neuroscience. 5 (1), 45-50 (2003).</li><li>Bloomquist, J. R., Roush, R. T., ffrench-Constant, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+neuronal+sensitivity+to+dieldrin+and+picrotoxinin+in+a+cyclodiene-resistant+strain+of+Drosophila+melanogaster+(Meigen).">Reduced neuronal sensitivity to dieldrin and picrotoxinin in a cyclodiene-resistant strain of Drosophila melanogaster (Meigen).</a> Archives of Insect Biochemistry and Physiology. 19 (1), 17-25 (1992).</li><li>Mutunga, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxicology+of+bis(n)-tacrines+on+Blattella+germanica+and+Drosophila+melanogaster+acetylcholinesterase.">Neurotoxicology of bis(n)-tacrines on Blattella germanica and Drosophila melanogaster acetylcholinesterase.</a> Archives of Insect Biochemistry and Physiology. 83 (4), 180-194 (2013).</li><li>Chen, R., Swale, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inwardly+rectifying+potassium+(Kir)+channels+represent+a+critical+ion+conductance+pathway+in+the+nervous+systems+of+insects.">Inwardly rectifying potassium (Kir) channels represent a critical ion conductance pathway in the nervous systems of insects.</a> Scientific Reports. 8 (1), 1617 (2018).</li><li>Francis, S. A., Taylor-Wells, J., Gross, A. D., Bloomquist, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+and+physiological+actions+of+carbonic+anhydrase+inhibitors+to+Aedes+aegypti+and+Drosophila+melanogaster.">Toxicity and physiological actions of carbonic anhydrase inhibitors to Aedes aegypti and Drosophila melanogaster.</a> Insects. 8 (1), 2 (2016).</li><li>Swale, D. 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The details provided in the associated video and text have provided key steps in order to record the activity and spike discharge frequency of the Drosophila CNS ex vivo. The dissection efficacy is the most critical aspect of the method because short or few descending neurons will reduce the baseline firing rate that will result in large variances between replicates. However, once the dissection technique has been mastered, the data collected with this assay are highly reproducible and amendable for a w...

The overall goal of this approach is to enable researchers to quickly measure the electrogenesis of the central nervous system (CNS) in the model insect, Drosophila melanogaster. This method is reliable, quick, and cost-efficient to perform physiological and toxicological experimentation. The CNS is essential for life and therefore, the physiological pathways critical for proper neural function have been explored extensively in an effort to understand or modify neural function. Characterization of the signaling pathways within the arthropod CNS has enabled the discovery of several chemical classes of insecticides that disrupt invertebrate neural function to induce mortality while limiting off-target consequences. Thus, the ability to measure the neural activity of insects is of significant interest to the field of insect toxicology and physiology since the nervous system is the target tissue of the majority of deployed insecticides1. Yet, continued growth of fundamental and applied knowledge regarding the insect nervous system requires advanced neurophysiological techniques that are limited in feasibility, since current techniques are labor intensive and require a high expense, insect neural cell lines are limited, and/or there is limited access to the central synapses of most arthropods. Currently, characterization of most insect neural proteins requires the target to be cloned and heterologously expressed for subsequent drug discovery and electrophysiological recordings, as was described for insect inward rectifier potassium channels2, insect ryanodine receptor3, mosquito voltage-sensitive K+ channels4, and others. To mitigate the requirement for heterologous expression and the potential for low functional expression, Bloomquist and colleagues aimed to induce a neuronal phenotype in cultured Spodoptera frugiperda (Sf21) cells as a novel method for insecticide discovery5,6. These methods provide a valid approach for the development of new chemistry, yet they oftentimes create an insurmountable bottleneck for the characterization of pharmacological agents, identifying mechanisms of insecticide resistance, and characterization of fundamental physiological principles. Here, we describe an ex vivo method that enables the recording of electrical activity from a model insect that has malleable genetics7,8,9 and known expression patterns of neural complexes10,11,12 to enable the characterization of resistance mechanisms at the level of the nerve, the mode of action of newly developed drugs, and other toxicological studies.
The fruit fly, D. melanogaster, is a common model organism for defining insect neural systems or insecticide mechanism of action and has been established as a well-suited model organism for the study of toxicological13, pharmacological14,15, neurophysiological16, and pathophysiological17,18,19,20 processes of vertebrates. D.melanogaster is a holometabolous insect that performs complete metamorphosis, including a larval and pupal stage before reaching the reproductive adult stage. Throughout the developmental process, the nervous system undergoes significant remodeling at different life stages, but the larval CNS will be the focus of this methodology. The fully developed larval CNS is anatomically simple with thoracic and abdominal segments that are fused and form the ventral ganglion, which represents an array of repeated and almost identical neuromeric units21,22. Descending motor nerves originate from the caudal end of the subesophageal ganglia and descend to innervate body wall muscles and visceral organs of the larvae. Figure 1 describes the gross anatomy of the larval Drosophila CNS.
The Drosophila blood-brain barrier (BBB) develops at the end of embryogenesis and is formed by subperineurial glial cells (SPG)21. The SPG cells form numerous filopodia-like processes that spread out to establish a contiguous, very flat, endothelial-like sheet that covers the entire Drosophila CNS23. The Drosophila BBB has similarities to the vertebrate BBB, which includes preserving the homeostasis of the neural microenvironment by controlling the entry of nutrients and xenobiotics into the CNS21. This is a prerequisite for reliable neural transmission and function, yet the protection of the CNS by the BBB restricts the permeation of synthetic drugs, most peptides, and other xenobiotics24,25, which introduces potential problems when characterizing potencies of small-molecule modulators. The method uses a simple transection to disrupt this barrier and provide ready pharmacological access to the central synapses. 
The greatest strength of the described methodology is the simplicity, reproducibility, and relatively high-throughput capacity inherent to this system. The protocol is relatively easy to master, the setup requires little space, and only an initial financial input is necessary which is reduced to reagents and consumables. Further, the described method is completely amendable to record the central descending nerve activity of the house fly, Musca domestica26.

<table>
	<tbody>
		<tr>
			<td>Drosophila melanogaster (strain OR)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>2376</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Kinetic Systems</td>
			<td>9200 series</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Kinetic Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope on a Boom</td>
			<td>Nikon</td>
			<td>SMZ800N</td>
			<td>Multiple scopes can be used; boom stand is critical</td>
		</tr>
		<tr>
			<td>AC/DC differential amplifier</td>
			<td>ADInstruments</td>
			<td>AM3000H</td>
			<td>The model 1700 can be used instead of the model 3000</td>
		</tr>
		<tr>
			<td>audio monitor</td>
			<td>ADInstruments</td>
			<td>AM3300</td>
			<td></td>
		</tr>
		<tr>
			<td>Hum Bug Noise Eliminator</td>
			<td>A-M Systems</td>
			<td>726300</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition System (PowerLab)</td>
			<td>ADInstruments</td>
			<td>PL3504</td>
			<td>Multiple PowerLab models can be used.</td>
		</tr>
		<tr>
			<td>Lab Chart Pro Software</td>
			<td>ADInstruments</td>
			<td>N/A - Online Download</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Optic Lights</td>
			<td>Edmund Optics</td>
			<td>89-740</td>
			<td>Different light sources can be used, but fiber optics are the most adaptable</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M325</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Holder</td>
			<td>World Precision Instruments</td>
			<td>MEH715</td>
			<td>Different models are acceptable</td>
		</tr>
		<tr>
			<td>BNC cables</td>
			<td>World Precision Instruments</td>
			<td>multiple based on size</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>PG52151-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td>Also can use Narashige PC-100</td>
		</tr>
		<tr>
			<td>Black Wax</td>
			<td>Carolina Biological Supply</td>
			<td>974228</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-coated insect pins, size #2</td>
			<td>Bioquip</td>
			<td>1208S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fince Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophysiological recording of the central nervous system activity of third-instar drosophila melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Spontaneous activity of the descending peripheral nerves arising from the Drosophila central nervous system can be recorded using extracellular suction electrodes with consistent reproducibility. Spontaneous activity of the excised and transected Drosophila CNS produces a cyclical pattern of bursting with 1-2 s of firing with approximately 1 s of near quiescent activity. For example, the CNS is near quiescent (1-2 Hz) for 0.5-1 s, followed by a burst (100-400 Hz) for app...

Watch this Scientific Journal Video about Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster at JoVE.com

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

This method can help answer key questions in the fields of insect toxicology and insect physiology by measuring the electrogenesis of the Drosophila melanogaster central nervous system. This allows one to test a wide range of scientific hypotheses and aid in the discovery of new insecticide modes of action. The main advantage of this technique is that it provides a simple, reproducible, and relatively high-throughput system with minimal financial input to study the nervous system of Drosophila.To start, open the acquisition/analysis software. Click Setup in the main toolbar, and select Channel Settings, which will open a dialog box. Reduce the number of total channels to three.Channel one will record the raw signal from the amplifier with a range of 100 millivolts. Channel two will be used to count the number of events from channel one that are above a given threshold. For this, click on the Calculation tab to open a drop-down menu for channel two.Select Cyclic Measurements, which will open a second dialog box. Select channel one as the source. Then, on the drop-down menu measurement, select Unit Spike at Events.Finally, in the detection settings area, from the drop-down menu, select Simple Threshold. The threshold level is input on the detection adjustment window to be set above background noise. To convert the electrical activity into a rate plot expressed in hertz, select Arithmetic on the third channel.On the new window, input 1000 times smoothsec, parentheses channel two comma two. Then, select at the units drop-down menu the frequency unit hertz. Close the dialog boxes to return to the main screen.The y-axis of channel three should be expressed in hertz and the x-axis in time. Dissect the larval Drosophila CNS by first identifying and extracting a wandering third-instar Drosophila melanogaster from the culture vial and placing it into 200 microliters of saline. Then, grasp the mouth hooks with a pair of fine forceps, and grab the abdomen of the maggot with a second pair of forceps, with light pressure, to avoid tearing the thin cuticular layer.Gently pull the mouth hooks and abdomen in different directions to separate the caudal end of the maggot from the head region and expose the viscera of the maggot. The CNS will be intertwined with the trachea and digestive tract. Tease the CNS out of the digestive tract and trachea with forceps.The dissection of the Drosophila central nervous system from the maggot is a critical step for success. The dissected ganglia must be intact, without any significant damage to the descending motor nerves. A larger number of motor nerves attached to the ganglia will increase baseline firing frequencies.If necessary, disrupt the blood-brain barrier by manually transecting the CNS posterior to the cerebral lobes with Vannas spring scissors. The transection should be done based on the physiochemical properties of the chemical being used. The red line is the suggested transection point and the transected CNS with intact descending peripheral nerve trunks shown here.To begin, first pull the glass pipette electrode from borosilicate glass capillaries to a resistance of five to 15 megaohms. Insert the transected CNS into a wax chamber containing 200 microliters of saline. Clamp an uncoated insect pin with an alligator clip soldered to the ground wire, and insert the pin into the saline to complete the circuit.Using the micromanipulators, orient the electrode to the caudal end of the transected CNS. Eliminate background noise by adjusting the threshold level in the acquisition/analysis software prior to connecting the peripheral nerve trunks. Apply slight negative pressure on the syringe to draw peripheral nerves into the suction electrode.Start the recording on the data acquisition software, and the baseline firing rate to equilibrate for five minutes prior to collecting baseline firing rate data. After five minutes, add 200 microliters of saline and vehicle to bring the total volume of the chamber to 400 microliters to begin recording control firing rates. Discard the preparation and recording if the pattern of firing of control treatment is not similar to the example shown here.When baseline has been established after three to five minutes of recording, withdraw 200 microliters of saline and add 200 microliters of the experimental agent solubilized in saline. Label this time point of drug application in the acquisition/analysis software by including a comment that includes the drug and the final concentration. Shown here is a representative nerve discharge trace before and after exposure to DMSO.The arrow indicates the time of application. A limited response to DMSO is seen. Increasing doses of propoxur amplified the spike discharge frequency of the transected Drosophila CNS in a concentration-dependent manner, whereas the neurodepressant GABA reduced the spike discharge frequency in a concentration-dependent manner.While attempting this procedure, it's important to remember that it is an ex vivo experiment, and therefore the conditions of the saline, such as pH and temperature, is critical for prolonged activity of the central nervous system preparation. Similarly, the lack of extraneous electrical activity, an effective Faraday cage, and a reduction of 60-hertz noise are critical for success of this assay. The data collected through this assay can help identify the specific mode of action of insecticides.Subsequent validation of these data through other methods such as voltage-clamp electrophysiology, biochemical analyses, and additional pharmacological assays can be performed.

Research

JoVE Journal

Neuroscience

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