title: Paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the Drosophila melanogaster giant fiber system.
abstract: Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents. 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 melanogaster. 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. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on Dα7 nicotinic acetylcholine receptors (nAChRs). Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System 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 al, 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.
authors.affiliation: Department of Biological Sciences, Florida Atlantic University, FL, USA.
authors.forename: Monica
authors.initials: M
authors.lastname: Mejia
authors.forename: Mari D
authors.initials: MD
authors.lastname: Heghinian
authors.forename: Alexandra
authors.initials: A
authors.lastname: Busch
authors.forename: Frank
authors.initials: F
authors.lastname: Marí
authors.forename: Tanja A
authors.initials: TA
authors.lastname: Godenschwege
mesh_terms: D000157:Aconitine / Q000008:administration & dosage / Q000031:analogs & derivatives; D000818:Animals; D002951:Citrates / Q000008:administration & dosage; D004331:Drosophila melanogaster / Q000502:physiology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D007306:Insecticides; D036103:Nanotechnology / Q000379:methods*; D009412:Nerve Fibers / Q000187:drug effects* / Q000502:physiology*; D018733:Nicotinic Antagonists / Q000008:administration & dosage
chemical_list: D002951:Citrates; D007306:Insecticides; D018733:Nicotinic Antagonists; C054634:methyllycaconitine; D000157:Aconitine