Steroid hormones can modulate motivated behaviors through the mesocorticolimbic system. Gonadectomy (GDX) is a common method to determine how steroids influence the mesocorticolimbic system, and caloric restriction (CR) is often used to invigorate motivated behaviors. A common assumption is that the effects of these manipulations on brain steroid levels reflects circulating steroid levels. We now know that the brain regulates local steroid levels in a region-specific manner; however, previous studies have low spatial resolution. Using ultrasensitive liquid chromatography tandem mass spectrometry, we examined steroids in microdissected regions of the mesocorticolimbic system (ventral tegmental area, nucleus accumbens, medial prefrontal cortex). We examined whether GDX or CR influences systemic and local steroids, particularly testosterone (T) and steroidogenic enzyme transcripts. Adult male rats underwent a GDX surgery and/or CR for either 2 or 6 weeks. Levels of T, the primary steroid of interest, were higher in all brain regions than in the blood, whereas corticosterone (CORT) was lower in the brain than in the blood. Importantly, GDX completely eliminated T in the blood and lowered T in the brain. Yet, T remained present in the brain, even 6 weeks after GDX. CR decreased both T and CORT in the blood and brain. Steroidogenic enzyme (Cyp17a1, 3β-hydroxysteroid dehydrogenase, aromatase) transcripts and androgen receptor transcripts were expressed in the mesocorticolimbic system and differentially affected by GDX and CR. Together, these results suggest that T is synthesized within the mesocorticolimbic system. These results provide a foundation for future studies examining how neurosteroids influence behaviors mediated by the mesocorticolimbic system.

Steroids are secreted by the gonads and adrenal glands into the blood to modulate neurophysiology and behaviour. In addition, the brain can metabolise circulating steroids and synthesise steroids de novo. Songbirds show high levels of neurosteroid synthesis. In the present study, we developed and validated a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) assay for the measurement of 10 steroids in whole blood, plasma and microdissected brain tissue (1‐2 mg) of song sparrows. Our assay is highly accurate, precise, specific and sensitive. Moreover, the liquid‐liquid extraction is fast, simple and effective. We quantified steroids in the blood and brain of wild male song sparrows in both breeding and non‐breeding seasons. As expected, systemic androgen levels were higher in the breeding season than in the non‐breeding season. Brain androgens were detectable only in the breeding season; androstenedione and 5α‐dihydrotestosterone levels were up to 20‐fold higher in specific brain regions than in blood. Oestrogens were not detectable in blood in both seasons. Oestrone and 17β‐oestradiol were detectable in brain in the breeding season only (up to 1.4 ng g‐1 combined). Progesterone levels in several regions were higher in the non‐breeding season than the breeding season, despite the lack of seasonal changes in systemic progesterone. Corticosterone levels in the blood were higher in the breeding season than in the non‐breeding season but showed few seasonal differences in the brain. In general, the steroid levels presented here are lower than those in previous reports using immunoassays, because of the higher specificity of mass spectrometry. We conclude that (i) brain steroid levels can differ greatly from circulating steroid levels and (ii) brain steroid levels show region‐specific seasonal patterns that are not a simple reflection of circulating steroid levels. This approach using ultrasensitive LC‐MS/MS is broadly applicable to other species and allows steroid profiling in microdissected brain regions.

The white-throated sparrow (Zonotrichia albicollis) represents a powerful model in behavioral neuroendocrinology because it occurs in two plumage morphs that differ with respect to steroid-dependent social behaviors. Birds of the white-striped (WS) morph engage in more territorial aggression than do birds of the tan-striped (TS) morph, and the TS birds engage in more parenting behavior. This behavioral polymorphism is caused by a chromosomal inversion that has captured many genes, including estrogen receptor alpha (ERα). In this study, we tested the hypothesis that morph differences in aggression might be explained by differential sensitivity to estradiol (E2). We administered E2 non-invasively to non-breeding white-throated sparrows and quantified aggression toward a conspecific 10 min later. E2 administration rapidly increased aggression in WS birds but not TS birds, consistent with our hypothesis that differential sensitivity to E2 may at least partially explain morph differences in aggression. To query the site of E2 action in the brain, we administered E2 and quantified Egr-1 expression in brain regions in which expression of ERα is known to differ between the morphs. E2 treatment decreased Egr-1 immunoreactivity in nucleus taeniae of the amygdala, but this effect did not depend on morph. Overall, our results support a role for differential effects of E2 on aggression in the two morphs, but more research will be needed to determine the neuroanatomical site of action.

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