Model p 97

Printing nozzles were fabricated by pulling borosilicate capillaries (World Precision Instruments TW100‐4) to an outer diameter of ~2–3 µm (Sutter Model P‐97). The capillaries were then rendered conductive by a thin coating of Ti/Au (5 and 50 nm) via e-beam evaporation. To print, the nozzles are back-filled with an ink and moved to a working distance ~10 μm from the substrate. The sample is positioned on a conductive grounded plate and electrohydrodynamic ejection is induced by applying a square wave voltage of 250–350 V_p at 1 kHz to the nozzle. All printing was performed under ambient conditions (23 °C, 30–45% relative humidity). The printing setup used for this work is equipped with a confocal laser microscope for in-situ visual inspection. The pulse length, voltage applied to the nozzle, and stage position are controlled using a custom-built control unit.

Electroantennograms (EAG) were obtained by inserting a saline-filled sharp glass electrode (resistance ~ 10 MΩ) into the antenna and a silver-chloride ground electrode into one eye and were recorded by a DC amplifier (Model 440; Brown-Lee Precision, San Jose, CA). To make LFP and patch clamp recordings, a wax cup was built around the head to hold a bath of locust ringer solution, and a small section of the cuticle was then removed to expose the brain^{26 (link)}. To record LFPs, a blunt glass, saline-filled electrode was placed above the calyx of the mushroom body. For whole-cell patch clamp recordings, patch electrodes were pulled from borosilicate glass capillaries by a pipette puller (Model P-97; Sutter Instruments), with the program parameters tuned to produce an electrode resistance around 6 MΩ when filled with locust internal solution^{27 (link)} that was adjusted to osmolarity of about 350. Data were recorded through a MultiClamp 700A microelectrode amplifier and digitized via Digidata 1322A. Stimulus delivery and recordings were controlled by our custom LabView program running on a data acquisition PC.
EAG recordings and patch clamp recordings from PNs and LNs were obtained from 6 consecutive trials with each odor, and LFP responses were recorded in 10 trials with each odor. All experiments were conducted on awake animals.

Wild-type NHGRI-1 fish were bred and maintained using standard procedures (LaFave et al., 2014 (link)). Embryos were obtained by natural spawning and staged as described (Kimmel et al., 1995 (link)). All zebrafish works were approved by the Institutional Animal Care and Use Committee, Office of Animal Welfare Assurance, University of California, Davis.
In vitro transcribed capped RNAs were prepared using the mMessage mMachine RNA Synthesis Kit (AM1340, Thermo Fisher Scientific) and purified using the RNeasy Mini Kit (74104, Qiagen, Germantown, MD) following manufacturers' instructions.
Microinjection of RNA was performed as described (Jao et al., 2012 (link)). In brief, one-cell-stage embryos from wild-type zebrafish intercrosses were injected with the following amounts of in vitro transcribed RNA: Cas9, 400 pg; Kif26b, 400 pg; Wnt5a, 150 pg. Pipettes were pulled on a micropipette puller (Model P-97, Sutter Instruments, Novato, CA). Injections were performed with an air injection apparatus (Pneumatic MPPI-2 Pressure Injector, Eugene, OR). Injected volume (typically ~1 nl) was calibrated with a microruler.

Secondary follicles collected from four mice were combined and randomly distributed to the negative control siRNA and Figla siRNA injection groups, and siRNA was microinjected into the oocytes of secondary follicles as previously reported⁵⁴ . Specifically, intact follicles were placed in M2 medium (M-7167, Sigma-Aldrich) droplets prepared in a microinjection chamber. Injection needles were prepared using a puller (Model P-97, Sutter instrument, US) with the following protocol: heat = 655, pull = 85, vel = 120, del = 110, pressure 300. An injection needle was filled with siRNA reagent and placed on a manipulator (ONM-2D, Narishige, Japan) set on an inverted microscope (IX71, Olympus, Japan). Follicles were held, the injection needle penetrated the cytoplasm of the oocyte, and siRNA reagent was injected with one shot of the FemtoJet (Eppendorf, Germany). After the microinjection, 12–13 follicles were placed on collagen-coated inserts (Transwell COL #3493, Corning, US) set in 12-well plates containing 2 ml of IVG medium in each well and incubated for 12 days (37 °C, 5% CO₂) as previously described^{55 (link)} with slight modifications. The medium around the filter was changed every 4 days.

Two-electrode voltage-clamp electrophysiology was used to record currents produced by activation of the expressed Bma-SLO-1 channels. Recordings from water injected oocytes served as control experiments. Recordings were made using an Axoclamp 2B amplifier (Warner Instruments, USA) with the oocytes voltage-clamped at +20 mV, and data acquired on a computer with Clampex 10.3 (Molecular Devices, CA, USA). The microelectrodes used to impale the oocytes were pulled using a Flaming/Brown horizontal electrode puller (Model P-97, Sutter Instruments, USA) set to pull micropipettes that when filled with 3 M KCl had a resistance of 20–30 MΩ. The micropipettes tips were carefully broken with a piece of tissue paper in order to achieve a resistance of 2–5 MΩ in recording solution (100 mM NaCl, 2.5 mM KCl, 1 mM CaCl₂.2H₂O and 5 mM HEPES, pH 7.3). The low resistance pipettes allowed large currents to be passed to maintain adequate voltage-clamp.
Emodepside used in this study was obtained from Bayer Animal Health. Potassium channel inhibitor iberiotoxin from Sigma Aldrich (St. Louis, MO, USA). The drugs were solubilized in DMSO and diluted in recording solution.