Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the <em>Drosophila melanogaster</em> 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 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 Dα7 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.

Transforming conotoxins into cyclotides: Backbone cyclization of P‐superfamily conotoxins

“…Linear and cyclic gm9a were tested on the Drosophila GFS using the paired electrophysiology/nanoinjection bioassays as described previously . P[Gaw‐ B]OK307 (Stock #6488; Bloomington Stock Center, Bloomington, IN; referred to as A307 henceforth) fly stocks were kept at either 22°C or 25°C in vials containing standard media.…”

Inhibition of cholinergic pathways inDrosophila melanogasterby α‐conotoxins

“…We used the Drosophila GF assay (10, 15) to screen venom fractions from C. brunneus , an Eastern Pacific worm‐hunting cone snail species. The HPLC fraction eluting at ~40 min (Fig.…”

“…Female gfA PΔEY6 ;A307 flies were crossed to UAS‐Dα7, UAS‐Dα7‐D77T/L117Q/I196P and reared at 25°C. Male offspring was tested using the paired electrophysiology/nanoinjection bioassay described previously (10, 15). BruIB, PelA, BulA, and Iml were injected at 45 pmol/fly ( n = 10 for each peptide), and flies were evaluated for changes in the FF of the GF‐DLM and GF‐TTM pathway at 50 Hz in the UAS‐Dα7‐D77T/L117Q/I196P line.…”

“…α‐Conotoxins were tested using the D. melanogaster GFS with the paired electrophysiology/nanoinjection bioassay as described previously (10, 15). P[Gaw‐ B]OK307 (Stock #6488; Bloomington Stock Center, Bloomington, IN, USA; referred to as A307 henceforth) fly stocks were kept at either 22°C or 25°C in vials containing standard media.…”