P 97 puller

Electrophysiological recordings were performed using a two-electrode voltage clamp and a GeneClamp 500 amplifier (Molecular Devices, Union City, CA, USA). Oocytes were immersed in BAMS or OR₂ mediums and impaled with 2 intracellular glass electrodes filled with 3M CsCl or 3 M KCl. The resistance of the pulled electrodes (P-97 puller; Sutter Instruments, Novato, CA, USA) was 1–2 MΩ. The perfusion system was controlled by a Manifold Solution Changer (MSC-200; Bio-Logic; Grenoble, France). Data acquisition and analysis were performed using Clampex and Clampfit from Pclamp10 software (Molecular Devices, Union City, CA, USA). Leak and capacitive currents were subtracted during analysis. The study of the effects of the different samples (ATX, SFE-Extract, SFE-Extract + ATX) was carried out on Xenopus oocytes recorded endogenous currents using 250 ms/300 ms or 500 ms depolarizing pulses from −60 to +120 mV at holding potentials of −80 or −60 mV.

Whole-cell ion currents were experimentally recorded with an arrangement of Axopatch 200B/Digidata 1550/pCamp10 (amplifier/analog-digital converter/software, all from Molecular Devices, Sunnyvale, CA), analog filtered at 5 kHz and digitally sampled at 10 kHz. Patch-clamp pipettes were made of borosilicate glass (World Precision Instrument) using a P-97 puller (Sutter Instruments, Novato, CA). The intracellular pipette filling solution was composed of (in mM) 10 NaCl, 40 KCl, 10 HEPES, 5 EGTA, 3 MgCl₂, 95 K-gluconate, and 10 glucose (pH 7.2 adjusted with KOH). The extracellular solution contained (in mM) 140 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, and 5 glucose (pH 7.4 adjusted with NaOH). All solutions were perfused using a custom-made gravity-based perfusion system. The voltage-clamp protocol used in all experiments was 200 ms step pulses from -120 to +50 mV in 10 mV increments from a holding voltage of -70 mV.

Electrophysiological recording was performed in whole-cell patch clamping mode by using HEKA EPC10 amplifier as described previously (Gong et al., 2016 (link)). Micropipettes were pulled from borosilicate glass capillary tubes (Word Precision Instruments) by using P-97 puller (Sutter instruments). The micropipette solution contained 120 mM CsCl, 10 mM HEPES, 10 mM EGTA, 0.3 mM Na-GTP, 3 mM Mg-ATP. Adjust the pH to 7.2–7.4 with CsOH, and adjust the osmotic pressure with dd H₂O to about 305. Add 5 mM QX-314 to the micropipette solution before use for the evoke release recording. The bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose. Adjust the pH to 7.2–7.4 with NaOH, and adjust the osmotic pressure with dd H₂O to about 315. Inhibitory postsynaptic currents (IPSCs) were isolated by adding the AMPA and NMDA receptor blockers CNQX (20 μM) and AP-5 (50 μM) to the bath solution. Evoked IPSC was recorded in the stimulus pulse (90 μA) mini IPSCs were recorded in the presence of tetrodotoxin (1 μM) for action-potential blocking. Sucrose IPSC was measured with a 0.5 M sucrose application in the bath solution. Electrophysiological data analyzed by Clampfit 10 (Molecular devices).

Thirty-five wild-caught dragonflies (35 Hemicordulia tau, 33 male, two female) were immobilized with 1:1 beeswax and rosin mixture and fixed to an articulated magnetic stand with the head tilted forward to access the posterior surface. A hole was cut above the brain to gain access to the lobula and lateral midbrain, but the preparation was otherwise left with the perineural sheath and overlying haemolymph sacs intact. All dissections were performed on the left side of the animal corresponding to a right excitatory hemifield. We penetrated the sheath and recorded intracellularly using aluminosilicate micropipettes (OD = 1.00, ID = 0.58 mm), pulled on a Sutter Instruments P-97 puller and backfilled with either KCl (2 M, electrode tip resistance typically 50–150 MΩ) or 4% Lucifer Yellow solution in 0.1 M LiCl. Electrodes were placed in the medial portion of the lobula complex and stepped through the brain from posterior to anterior through the lobula complex, using a piezoelectric stepper (Marzhauser-Wetzlar PM-10, Wetzlar, Germany). Intracellular responses were digitized at 5 kHz with a 16-bit A/D converter (National Instruments) for offline analysis.