DOI: 10.26190/unsworks/31718
title: Sodium Butyrate Rescues Neurodevelopmental Deficits Following Perinatal Methadone Exposure - pup allocation information

description: Detailed information about pup experimental allocation for the paper Sodium Butyrate Rescues Neurodevelopmental Deficits Following Perinatal Methadone Exposure - under review The first tab "Dams" contains dam level information - the dams' ID in column A give each unique dam ID, where the first number denotes the cohort the dam was in and the second the dams randomly assigned number within that cohort. For example, DamID 1.2 indicates a dam in cohort 1 that was assigned to be number 2. These IDs link the pup level data in tab two to each dam and is part of a unique identification code for each pup, linking them to the pup level data in tab two "Pups". The allocation of dams to experimental groups is in column B. The number of male and female pups left to each dam after the pups were culled in columns C and D. Column E lists experimental outcomes that utilised all the pups in the litter and how that data was treated. The second tab, "Pups" contains data about which individual pups from each litter were allocated to each experimental outcome. Column A contains information about if each pup received methadone "Met" or control surgery "Sham", column B about whether they received sodium butyrate "NaB" or water "Veh". Column C links to the dam ID in tab one. The same DamID in colomn C denotes pup that come from the same litter. Coloum E represents pup's ID, where the first number represents the dam that the pup came from, and the second number is a number that was randomly allocated to that pup within its litter. For example, PupID 2.1 indicated that the pup from dam 2. Together, the DamID and pupID provide unique identification for each pup. Column F represents the experimental outcomes each pup was used for.
description: Twenty-six time-mated female Sprague Dawley rats (223-332g; Animal Resource Centre, AU) were purchased at gestational day 3 (GD3) and maintained on a 12-hour light cycle (on at 0700h) in a temperature- and humidity-controlled colony room (22°C±1) with ad libitum access to food. All dams gave birth within 24 hours of GD22 (postnatal day 0) and litters were culled to 10 (5 male, 5 female where possible). On P22 pups were weaned into cages of 2-5 rats, matching for sex and experimental condition. Methadone hydrochloride (National Institute of Measurement, NSW, AU) was dissolved in sterile saline and administered to dams via mini osmotic pumps (2ML2, Alzet CA, USA) at 9 mg/kg/day from GD10 until P17 (Fig 1A). Minipumps were used to minimize the impact on the dams. This dose produces stable concentration in dam blood, accumulation in pup brain, and physiologically-relevant concentrations in pup urine. Sodium butyrate (Sigma-Aldrich, Missouri, USA) was administered via drinking water (3% w/v, refreshed every 48-72 hours) across GD10-P12. This concentration is well tolerated by rats and results in increased fecal levels of butyric acid. This period of administration was chosen to mimic treatment during prenatal development in human infants, as rats are born earlier in the developmental process than human infants, thus assessing the impact of treatment with NaB via the mother during gestion without direct treatment of the offspring. On GD10 dams were randomly allocated to groups: sham surgery/water (Sham/Veh), methadone pump/water (Met/Veh), sham surgery/sodium butyrate (Sham/NaB) and methadone pump/sodium butyrate (Met/NaB). Mini-osmotic pump surgery was performed according to manufacturer’s instructions at GD10 and replaced at P3 to permit continuous methadone exposure across gestation and the early postnatal period (GD10 until P17; Fig 1A). Control animals received sham surgery as sham and saline-filled mini pumps insertion produce similar effects. Pups were randomly assigned to the experimental outcomes listed.