Neuroestrogens are synthesized within the brain and regulate social behavior, learning and memory, and cognition. In song sparrows, Melospiza melodia, 17β-estradiol (17β-E 2 ) promotes aggressive behavior, including during the non-breeding season when circulating steroid levels are low. Estrogens are challenging to measure because they are present at very low levels, and current techniques often lack the sensitivity required.Furthermore, current methods often focus on 17β-E 2 and disregard other estrogens.Here, we developed and validated a method to measure four estrogens (estrone, 17β-E 2 , 17α-estradiol, estriol) simultaneously in microdissected songbird brain, with high specificity, sensitivity, accuracy, and precision. We used liquid chromatography tandem mass spectrometry (LC-MS/MS), and to improve sensitivity, we derivatized estrogens using 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). The straightforward protocol improved sensitivity by 10-fold for some analytes. There is substantial regional variation in neuroestrogen levels in brain areas that regulate social behavior in male song sparrows. For example, the auditory area NCM, which has high aromatase levels, has the highest estrone and 17β-E 2 levels. In contrast, estrogen levels in blood are very low. Estrogen levels in both brain and circulation are lower in the non-breeding season than in the breeding season. This technique will be useful for estrogen measurement in songbirds and potentially other animal models.

Estrogens are steroid hormones that affect many aspects of brain function, including cognition, social behavior, and neuroprotection. It is well-known that estrogens are synthesized in the ovaries. Estrogens are also synthesized in the brain, where aromatase is expressed in specific regions. Importantly, estrogens play crucial roles in the brain, even at extremely low levels. Current assays lack the necessary sensitivity and/or specificity to measure brain-synthesized estrogens. Furthermore, current methods focus on only 17β-estradiol and generally disregard other estrogens that are synthesized in the brain. Here, we developed a method to measure several estrogens simultaneously, with high sensitivity and specificity. To improve sensitivity, we derivatized estrogens with 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to examine a panel of eight estrogens: 17β-estradiol, 17α-estradiol, estrone, estriol, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 4-methoxyestradiol. After derivatization, we have improved sensitivity 20-fold, detecting as little as 0.01 pg per sample, demonstrating that our method is extremely sensitive. For each analyte, we have identified a distinct retention time as well as 2 scheduled multiple reaction monitoring (sMRM) transitions that were used as quality control criteria for clear identification. Therefore, we are able to distinguish each estrogen (even stereoisomers) by the chromatographic separation and the sMRM, demonstrating that our method is highly specific. This method has been applied to microdissected brain samples. Initially, we used a songbird model because songbirds have high levels of aromatase and 17β-estradiol in specific brain regions. We were able to simultaneously quantify multiple estrogens in small amounts of brain sample (1-2 mg). We examined seasonal changes of estrogens in the brain and blood. Future work will apply this method to mouse, rat, and human samples and expand the panel of estrogens examined. Our ultra-sensitive assay is essential for small animal models, where estrogen measurement is extremely challenging because of the limited amount of brain tissue. This novel technique will also have wide-ranging applications for basic research and clinical testing, including estrogen measurement in humans with low estrogen levels, such as men, pre-pubertal children, and post-menopausal women.