*2.2. Animals and Tissue Preparation*

Animals, belonging to the long-lived strain MZM 04/10 were used at the following time points: 5 weeks post hatching (wph) (young-adult, age of the sexual maturity) and 27 wph (old animals). Animal maintenance was performed as previously described [26]. To avoid effects of circadian rhythms and feeding, animals were euthanized at 10:00 in a fasted state, with an overdose of anesthetics. They were placed for approximately 5–10 min in a solution containing 1 mg/mL in buffered ethyl 3-aminobenzoate methanesulfonate without prior sedation and observed until no vital signs (body and operculum movement, righting reflex) were observed.

For RNA extraction, 5 fish for each time point (5 and 27 wph) were decapitated, brains were rapidly dissected, kept in sterile tubes (Eppendorf BioPhotometer, Hamburg, Germany) with 500 μL of RNAlater (Qiagen, Hilden, Germany), and stored at 4 ◦C until the RNA extraction. For fluorescence in situ hybridization (FISH), 5 adult fish (at 20 wph) were decapitated, brains were rapidly excised and fixed in 4% paraformaldehyde (PFA)/PBS overnight (ON) at 4 ◦C. Then, brains were incubated in 20% sucrose solution ON at 4 ◦C and successively in 30% sucrose solution ON at 4 ◦C. Brains were then embedded in cryomount and frozen at −80 ◦C. Serial transverse and sagittal sections of 12 μm thickness were cut with a cryostat (Leica, Deerfield, IL, USA).
