In anticipation of an expected 97% loss between LPA reconstitution and injection, resulting from lipid interactions with pipettes, tubes, and syringe surfaces in the absence of lipid carrier proteins, the following protocol was developed to achieve a final injection concentration of LPA equivalent to 112 μM (fig. S1). Powdered 18:1 LPA (857130P, Avanti Polar Lipids) was dissolved in methanol, then aliquoted into low-retention tubes, vacuum-dried, and stored at −20°C. LPA was reconstituted by sonicating in Hanks’ balanced salt solution (HBSS; 14175-095, Gibco) for a 5 mM stock. Five microliters of this stock was administered intracranially. Following calculated losses and on the basis of an estimated CSF volume of 24.4 μl, this 112 μM injection concentration resulted in a working concentration of 18 μM in CSF or 1.8 μM in the whole brain. Control animals were injected with 5 μl of vehicle. P8 CSF and brain volumes were estimated on the basis of mathematical extrapolation of data presented by Chuang et al. (40).

For the final inhibitor experiment, powdered LPA was reconstituted in 0.1% fatty acid–free bovine serum albumin (BSA) in phosphate-buffered saline (PBS), aliquoted, and diluted to a 500 μM LPA stock in 0.01% BSA before injection. This resulted in an approximate concentration of 80 μM in CSF or 8 μM in whole brain. No losses were included in these calculations, as BSA significantly enhances LPA availability in aqueous solution.

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