Tw100f 4

Viral injections were performed as previously described (Beier et al., 2016 (link); Ma et al., 2020 (link)). Briefly, VSV and lentivirus stock was diluted to final working concentrations (described in the "Results" section) with DMEM (11995073; Fisher Scientific), with Fast Green dye (BP123-10; Fisher Scientific) as the injection marker. Glass capillaries (TW100F-4; World Precision Instruments) were pulled into injection needles with a pipette puller (P-97; Sutter Instruments), and the tips were trimmed to create a ~10 μm opening. The injection needle was mounted into a microelectrode holder connected to a pneumatic PicoPump (World Precision Instruments). For retina injection, 3 dpf larvae were anesthetized in Tricaine (0.013% w/v, AC118000500; Fisher Scientific) and mounted laterally inside the center chamber of a glass-bottom dish (P50G-1.5-14-F; MatTek) with 1.5% low melting-point agarose (BP1360; Fisher Scientific). 0.5 nl of virus solution was injected inside the temporal retina.

For chick neurons, microinjection pipettes (TW100F-4, World Precision Instruments, Inc. Sarasota, FL) were pulled on a Brown and Flaming horizontal pipette puller. For Aplysia neurons, microinjection pipettes (1B100F-4, World Precision Instruments) were pulled on a Narishige PP830 vertical pipette puller. Pipettes were then back-loaded with 1 mg/ml rhodamine tubulin (Cytoskeleton, Inc., Denver, CO, USA) in injection buffer (100 mM PIPES pH 7.0, 1 mM MgCl₂, 1 mM EGTA) as previously described⁸⁴ . Before injection, tubulin was thawed, spun at 13,000 g for 30 min, kept on ice and back-loaded into pipettes pre-chilled to 4 °C. Microinjection was performed using the NP2 micromanipulator and FemtoJet microinjection system (Eppendorf North America, New York, NY), visualized with a Nikon ECLIPSE TE2000 microscope using phase contrast optics with a 40x objective. Alternately, microinjection was performed using a Narishige hydraulic micromanipulator with injection pressure supplied from a 3 ml luer-lock syringe.

For proof of concept, sodium dihomo-gamma-linolenic acid was dissolved in water at a concentration of 10 μM. All other fatty acids and metabolites arrived in ethanol and were diluted to 1 or 2.5 μM with water, mock injections used 1% ethanol. A glass capillary tube (World Precision Instruments Inc., TW100F-4) was drawn into a needle, loaded, and used to inject into the gonad syncytium of the OD95 strain using an inverted light microscope. For each concentration of fatty acid metabolite, 8–12 individual nematodes were injected. Nematodes were recovered on NGM seeded with E. coli for 2 hours at 20˚C. Nematodes were then transferred to a drop of 0.1 mg/mL levamisole on a 2% agarose pad and a coverslip was applied. Germ cell membrane and nuclei were viewed with laser excitation at 488 and 561 nm (filtered at 495/535 nm and 600/670 nm for GFP and mCherry excitation/emission) and imaged on a Leica TCS SP5 confocal microscope with a 63X oil immersion lens. Images were processed identically using Adobe Photoshop CS4. The mean number of defective germ cells per injected worm was determined, and two-tailed student’s t test was used to compare the number of defective germ cells in injected worms compared to mock injections.

We performed intracellular single recordings on the mechanosensory T cells using sharp electrodes filled with 4 M potassium acetate (pH adjusted to 7.4). Electrode resistances ranged from 12 to 35 MΩ (mean = 24.26 MΩ, std = 5.06 MΩ). Electrodes were pulled from borosilicate thin-wall capillaries (TW100F-4, World Precision Instruments Inc., Sarasota, FL, United States) with a P97 Flaming Brown micropipette puller (Sutter Instruments Company, Novato, CA, United States). We performed the recordings using a mechanical micromanipulator type MX-1 (TR 1, Narishige, Tokyo, Japan) and a BA-1 s amplifier (NPI Electronic, Tamm, Germany). We identified the T cells according to their soma location and response patterns (Nicholls and Baylor, 1968 (link)). Current was injected into the soma while recording the membrane potential (sample rate 10 or 100 kHz; custom MATLAB software).