(R,S)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ((±)-CPP, Alomone Labs, C-175) and 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP hydrochloride, Abcam, ab120035) were prepared in distilled water and diluted with saline to the required concentration. Trans-N,N′-(Cyclohexane-1,4-diyl)bis(2-(4-chlorophenoxy) acetamide (ISRIB, Sigma, SML0843) was dissolved in dimethyl sulfoxide (DMSO) with gentle warming in a 40°C water bath and vortexed until the solution became clear. Then the solution was diluted in polyethylene glycol 400 (PEG400) with gentle warming in a 40°C water bath and vortexed. The solution was prepared freshly and diluted in warm saline (37°C) before injection. 1:1 DMSO and PEG400 in saline was used as vehicle control. The choice of dose and timing of ISRIB administration was based on previous reports [42 (link)] and our study of the pharmacokinetics of ISRIB in live rats [44 (link)]. Four pair-housed aged (17-18-month-old) rats received a single daily injection of ISRIB (2.5 mg/kg, i.p.) on 3 consecutive days. Then the rats stayed pair-housed for 18 days after the third injection of ISRIB.