Table 1.

Summary of the analysis approach and outcomes.

Analysis	Objective	Outcomes
1. Flexible factorial design with 2 groups (young and old) × 4 time points (baseline and three resting state scans). Comparisons between baseline and resting state 4.	To show age differences in ACC FC with the whole brain during DNIC.	Young group had increased ACC FC with the following network compared to old group (Fig. 3A): APT, RS, DpMe and Tg. Old group had increased ACC FC with the following network compared to young group (Fig. 3B): PAG, hippocampus, Rn and Pn.
2. Flexible factorial design with 2 groups (male and female) × 4 time points (baseline and three resting state scans). Comparisons between baseline and resting state 4.	To show sex differences in ACC FC with the whole brain during DNIC.	Males had increased ACC FC with the following network compared to females (Fig. 3C): PAG, hippocampus and DpMe. Females had increased ACC FC with the following network compared to males (Fig. 3D): RS, Pn, LC, PGi and Gi.
3. Flexible factorial design with 4 groups (YM, YF, OM and OF) and comparisons between baseline and resting state 4 for ACC FC.	To examine the full age-by-sex interaction in ACC FC with the whole brain during DNIC.	Effects of age in sex-matched groups YF had increased ACC FC with the following network compared to OF (Fig. 4A): RS, DpMe, Tg, Pt and cerebellum. OF had increased ACC FC with the following network compared to YF (Fig. 4B): PAG, hippocampus, Rn, Pn, and Sub. No differences between YM and OM. Effects of sex in age-matched groups YM had increased ACC FC with the following network compared to YF (Fig. 4C): PAG, hippocampus and DpMe. YF had increased ACC FC with the following network compared to YM (Fig. 4D): RS, Pn, LC, PGi, cerebellum and Pt. No differences between OM and OF.
4. Flexible factorial design with 4 groups (YM, YF, OM and OF) and comparisons between baseline and resting state 4 for PAG FC.	To examine the full age-by-sex interaction in PAG FC with the whole brain in old males during DNIC.	Effects of age in sex-matched groups OM had increased PAG FC with the following network compared to YM (Fig. 5A): Hippocampus, APT and RS. Effects of sex in age-matched groups OM had increased PAG FC with the following network compared to OF (Fig. 5B): Hippocampus, RS, Rn, Rt, Pn and LC.

Abbreviations: ACC: anterior cingulate cortex, APT: anterior pretectal nucleus, DpMe: deep mesencephalic nucleus, FC: functional connectivity, Gi: gigantocellular reticular nucleus, LC: locus coeruleus, OF: old female, OM: old male, PAG: periaqueductal gray, PGi: paragigantocellular nucleus, Pn: pontine nuclei, Pt: parietal association cortex, Rn: raphe nucleus, RS: retrosplenial cortex, Rt: pontine reticular nucleus, Sub: subiculum, Tg: tegmental area, YF: young female and YM: young male.