Standard 14 protein rodent diet

Nine male wild-type Sprague Dawley rats aged ca. 24 weeks (actual range 168-171 days) were used. Rats were group housed (3-4 per cage) in a SPF facility in open-top cages and maintained on a 12-12 h light-dark cycle (lights on at 07:00 h), under controlled temperature (22 ± 2 °C) and humidity (55 ± 5%) with free access to drinking water and standard 14% protein rodent diet (Harlan Teklad). Rats were killed by conscious decapitation and brains were immediately excised. Brain stem and cerebellum were removed and discarded. Fresh forebrains were weighed and pooled for homogenization before mitochondrial preparations.

Sprague-Dawley rats for the parental (P) generation were purchased from Charles River (Margate, Kent, UK). The first (F1) and second (F2) filial generations of control and prenatally stressed (PNS) offspring were bred in-house. Rats were maintained on a 12–12 h light–dark cycle, under controlled temperature (22 ± 2°C) and humidity (55 ± 5%) with free access to standard 14% protein rodent diet (Harlan Teklad). Breeding females were ad libitum fed a 50:50 mixture of 14% and 19% protein diet (Harlan Teklad) throughout pregnancy and lactation. Rats were group housed (4–6 females, 3–5 males) in open-top cages. A maximum of 2 rats/sex from each F2 litter were used/group for each experiment. All experiments were approved by the local Animal Welfare and Ethical Review Body and performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and the European Directive (2010/63/EU).

Female Sprague–Dawley rats were purchased from Charles River (Margate, UK). Unless otherwise specified, rats were group housed (four to six females, three or four males per cage) in open‐top cages and maintained under a 12 : 12 h light/dark cycle (lights on 07.00 h), at 22 ± 2 °C and 55 ± 5% relative humidity with free access to standard 14% protein rodent diet (Harlan Teklad, Derby, UK). All experiments were approved by the local Animal Welfare and Ethical Review Body and were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and the European Directive (2010/63/EU).
Control and PNS offspring were bred in‐house. Pregnant rats (approximately 12 weeks old) were obtained by overnight mating with sexually experienced males. Pregnancy was confirmed by the presence of a vaginal semen plug the next morning; this was designated as day 1 of pregnancy (parturition expected on day 22). The diet of breeding females was supplemented with 19% protein diet (Harlan Teklad) throughout pregnancy and lactation. Pregnant rats were initially group housed until day 14 of pregnancy, after which they were housed singly.