Specially formulated diets

Standard lab chow (LabDiet 5L0D, TX) containing approximately 70 ppm Mn (variable based on batch) was available ad libitum prior to dietary intervention. For dietary intervention mice were put on specially formulated diets (Research Diets, Inc.) that contained 70 ppm (control) or 2400 ppm Mn (high). Other micro- and macronutrient content was consistent between the two diets. The high Mn diet was chosen based previous reports that this Mn content significantly increased brain Mn after 30 days (Jenkitkasemwong et al., 2018 (link)). Diet was randomly assigned to a cage prior to behavioral testing.
Mice in the young experimental group were put on experimental diet at weaning (3 weeks of age; prior to genotyping) and were tested on a battery of behavioral tests at 11 weeks of age prior to sacrifice and dissection at 12 weeks of age (Fig. 1A). Mice in the aged experimental group were weaned at 3 weeks of age and were maintained on regular chow until a pre-intervention behavioral battery at approximately 11.5 months of age. Aged mice were then placed on experimental diet at 12 months of age, and 8 weeks later at approximately 14 months of age underwent a post-diet behavioral battery prior to sacrifice and dissection at approximately months of age (Fig. 2A).

Female Sprague-Dawley rats (n = 105; Charles River, Wilmington, MA) were kept under controlled conditions on a 12:12 light:dark cycle (21°C). To standardize competition for food and maternal attention, litters were reduced to 10–12 pups. At weaning (postnatal day (pnd) 21), rats were placed on specially formulated diets (Research Diets, New Brunswick, NJ). As weaning weight is known to affect age at puberty^{33 (link)}, only rats with weights within ±2 SD of the mean were used. Animals were weighed daily and monitored for vaginal opening from pnd 25. Group size was estimated using data from the effect of a high fat diet on puberty onset^{34 (link)}. Animals were euthanized on pnd 45, plasma collected and organ weights recorded. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Wyoming.

Body weight experiment: 53 animals were housed 3–6 per cage for the initial weight study.

Food Intake experiments: 24 animals were placed in individual cages for daily food intake.

Alternate diet experiments: 29 animals were placed in individual cages for daily food intake.

Fecal analysis and respiratory exchange ratio experiment: 28 rats were placed in individual metabolic cages that allowed for the collection of urine, fecal matter and recording of daily food intake.