Steroid profiling of glucocorticoids in microdissected mouse brain across development

Abstract

Corticosterone is produced by the adrenal glands and also produced locally by other organs, such as the brain. Local levels of corticosterone in specific brain regions during development are not known. Here, we microdissected brain tissue and developed a novel liquid chromatography tandem mass spectrometry method (LC‐MS/MS) to measure a panel of seven steroids (including 11‐deoxycorticosterone (DOC), corticosterone, and 11‐dehydrocorticosterone (DHC) in the blood, hippocampus (HPC), cerebral cortex (CC), and hypothalamus (HYP) of mice at postnatal day (PND) 5, 21, and 90. In a second cohort of mice, we measured the expression of three genes that code for steroidogenic enzymes that regulate corticosterone levels (Cyp11b1, Hsd11b1, and Hsd11b2) in the HPC, CC, and HYP. There were region‐specific patterns of steroid levels across development, including higher corticosterone levels in the HPC and HYP than in the blood at PND5. In contrast, corticosterone levels were higher in the blood than in all brain regions at PND21 and PND90. Brain corticosterone levels were not positively correlated with blood corticosterone levels, and correlations across brain regions increased with age. Local corticosterone levels were best predicted by local DOC levels at PND5, but by local DHC levels at PND21 and PND90. Transcripts for the three enzymes were detectable in all samples (with highest expression of Hsd11b1) and showed region‐specific changes with age. These data demonstrate that individual brain regions fine‐tune local levels of corticosterone during early development and that coupling of glucocorticoid levels across regions increases with age.

Glucocorticoid production in the thymus and brain: Immunosteroids and neurosteroids

“…While immunoassays typically measure one analyte per assay, mass spectrometry can simultaneously measure multiple analytes in a single sample for GC profiling. Microdissection of brain tissue (as little as 0.5 ​mg per sample) can be paired with LC-MS/MS to increase spatial specificity of GC profiling ( Hamden et al., 2021a ; Jalabert et al., 2021 ; Tobiansky et al., 2020 ). Measuring a panel of steroids in the GC synthesis pathway provides further insight into how tissues locally regulate GCs and their correlations (or lack thereof) with blood GCs.…”

“…Measuring a panel of steroids in the GC synthesis pathway provides further insight into how tissues locally regulate GCs and their correlations (or lack thereof) with blood GCs. For instance, a recent study measured a panel of GCs via LC-MS/MS, and brain corticosterone levels in different regions were more strongly correlated with brain 11-deoxycorticosterone (corticosterone precursor) and brain 11-dehydrocorticosterone (corticosterone metabolite), than with any blood GC levels ( Hamden et al., 2021a ). Additionally, mass spectrometry imaging can quantify GCs in tissue sections, with even greater spatial resolution ( Cobice et al., 2013 ).…”

“…During the SHRP, blood GC levels are very low and respond minimally to mild or moderate stressors. However, mice within the SHRP (postnatal day 2–12) ( Schmidt et al., 2003 ) locally upregulate corticosterone levels at baseline in lymphoid organs (e.g., thymus, bone marrow, spleen) and specific brain regions (e.g., hippocampus, hypothalamus), compared to the blood ( Hamden et al, 2019 , Hamden et al., 2021a ; Taves et al., 2015 ). Further, during the SHRP and adulthood, mouse brain corticosterone levels are more strongly correlated with GC levels within and between brain regions than with GC levels in the blood ( Hamden et al., 2021a ).…”

Sex-specific effects of neonatal oral sucrose treatment on growth and liver choline and glucocorticoid metabolism in adulthood

“…Steroids were extracted from 5 mL of serum, and progesterone, 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) as described previously (23). Calibration curves, blanks, and quality controls were processed alongside samples.…”

“…For steroid analyses, a value was considered nondetectable if the quantifier and qualifier transitions were not detected. For groups with less than 40% nondetectable values, steroid data were imputed using quantile regression imputation of left-censored missing data (23,29,30). Two-way analysis of variance (ANOVA) was used to assess the effects of sex and neonatal sucrose treatment.…”