Micromanipulator

For virus injections, we used adult wild-type mice (C57BL/6J). Animals were anesthetized with 10% ketamine (Bela-Pharm GmbH & Co. KG) and 2% xylazine (Rompun, Bayer Vital GmbH) in 0.9% NaCl (Fresenius). A volume of 1μl of the viral construct (AAV2.hSyn.iGluSnFR.WPRE.SV40, Penn Vector Core) was injected into the vitreous humour of both eyes via a Hamilton injection system (syringe: 7634-01, needles: 207434, point style 3, length 51mm, Hamilton Messtechnik GmbH) mounted on a micromanipulator (World Precision Instruments). Imaging experiments were performed 3 weeks after virus injection.

A 1450 nm diode laser (Capella, Lockheed Martin-Aculight, Bothell, Washington) coupled to a 400 µm core bare fiber (NA = 0.22; Ocean Optics, Dunedin, Florida) was used for all INS experiments. The optical fiber was positioned orthogonal to the nerve surface using a micromanipulator (World Precision Instruments, Sarasota, Florida). In accordance with previous optimization studies, diode current was adjusted to deliver radiant exposure between 1.4 and 1.6 J/cm² at a pulse width of 500 μs^{37 (link)}. The stimulation radiant exposure was determined by incrementally increasing the diode current until a muscle twitch was achieved for every delivered pulse. Pulse trains lasting 10 s at a repetition rate of 2 Hz were employed for every nerve monitoring trial to minimize thermal superposition^{58 (link)}. The optical fiber was positioned to not be in contact with the tissue at distance of ~ 120 μm such that the average spot size at the tissue was 503.6 ± 16 μm (1/e² diameter) as measured by an infrared beam profiler (BP209-IR2, Thorlabs, Newton, New Jersey) and validated using the knife-edge technique^{59 (link)}.

The viral construct AAV2.7m8.hSyn.iGluSnFR was generated in the Dalkara lab (for details, see ref. ^{74 (link)}). The iGluSnFR plasmid construct was provided by J. Marvin and L. Looger (Janelia Research Campus, USA). A volume of 1 μl of the viral construct was then injected into the vitreous humour of 3- to 8-week-old mice anaesthetized with 10% ketamine (Bela-Pharm GmbH & Co. KG) and 2% xylazine (Rompun, Bayer Vital GmbH) in 0.9% NaCl (Fresenius). For the injections, we used a micromanipulator (World Precision Instruments) and a Hamilton injection system (syringe: 7634-01, needles: 207434, point style 3, length 51 mm, Hamilton Messtechnik GmbH). Imaging experiments were performed 3–4 weeks after injection. As iGluSnFR expression tended to be weaker in the central retina, most OPL and IPL scan fields were acquired in the medial to peripheral ventral or dorsal retina.

Using a micromanipulator (World Precision Instruments, Sarasota, FL) to guide a positive displacement pipette (Drummond, scale 0.1–10 ul), a Nikon dissection microscope (4–10 X magnification) and untreated glass capillaries shaped using a micropipette puller (World Precision Instruments, Sarasota, FL) we microinjected 100 nl of normal saline (NS, 0.9% NaCl) or NS containing 20 ng human FGF23/ml in rapid sequence into the abdomen of flies immobilized on a CO₂ pad^{56 (link)}. Using this approach, it is possible to inject 20–30 flies within 1–2 minutes, which then were transferred into a clean culture tube with standard medium with or without mifepristone (R486). Pilot experiments showed very little lethality following microinjection and flies appeared to behave normally when observed for a week following the injection. However, flies were generally analyzed two hours after the injection as described below. Human FGF23 was provided by Susan Schiavi, Genzyme Inc. and tested on Western for integrity and able to induce hypophosphatemia in mice. FGF23 stocks were dissolved at 0.5 mg/ml 0.01 M acetic acid and stored at −70 °C prior to dilution to working stocks in NS. Assuming a total blood volume of flies of 2 ul injection of 100 nl of 20X FGF23 (20 ng/ml) will obtain a final concentration of 1 ng/ml, which 10-fold in excess of human or mouse concentrations of FGF23 (~0.1 ng/ml).