Chunqi Ma

Measurement of steroid concentrations in brain tissue: methodological considerations

It is well recognized that steroids are synthesized de novo in the brain (neurosteroids). In addition, steroids circulating in the blood enter the brain. Steroids play numerous roles in the brain, such as influencing neural development, adult neuroplasticity, behavior, neuroinflammation, and neurodegenerative diseases such as Alzheimer’s disease. In order to understand the regulation and functions of steroids in the brain, it is important to directly measure steroid concentrations in brain tissue. In this brief review, we discuss methods for the detection and quantification of steroids in the brain. We concisely present the major advantages and disadvantages of different technical approaches at various experimental stages: euthanasia, tissue collection, steroid extraction, steroid separation, and steroid measurement. We discuss, among other topics, the potential effects of anesthesia and saline perfusion prior to tissue collection; microdissection via Palkovits punch; solid phase extraction; chromatographic separation of steroids; and immunoassays and mass spectrometry for steroid quantification, particularly the use of mass spectrometry for “steroid profiling.” Finally, we discuss the interpretation of local steroid concentrations, such as comparing steroid levels in brain tissue with those in the circulation (plasma vs. whole blood samples; total vs. free steroid levels). We also present reference values for a variety of steroids in different brain regions of adult rats. This brief review highlights some of the major methodological considerations at multiple experimental stages and provides a broad framework for designing studies that examine local steroid levels in the brain as well as other steroidogenic tissues, such as thymus, breast, and prostate.

Inhibition of Hippocampal Aromatization Impairs Spatial Memory Performance in a Male Songbird

Recent studies have revealed the presence and regulation of aromatase at the vertebrate synapse, and identified a critical role played by presynaptic estradiol synthesis in the electrophysiological response to auditory and other social cues. However, if and how synaptic aromatization affects behavior remains to be directly tested. We have exploited 3 characteristics of the zebra finch hippocampus (HP) to test the role of synaptocrine estradiol provision on spatial memory function. Although the zebra finch HP contains abundant aromatase transcripts and enzyme activity, immunocytochemical studies reveal widespread pre- and postsynaptic, but sparse to undetectable somal, localization of this enzyme. Further, the superficial location of the avian HP makes possible the more exclusive manipulation of its neurochemical characteristics without perturbation of the neuropil and the resultant induction of astroglial aromatase. Last, as in other vertebrates, the HP is critical for spatial memory performance in this species. Here we report that local inhibition of hippocampal aromatization impairs spatial memory performance in an ecologically valid food-finding task. Local aromatase inhibition also resulted in lower levels of estradiol in the HP, but not in adjacent brain areas, and was achieved without the induction of astroglial aromatase. The observed decrement in acquisition and subsequent memory performance as a consequence of lowered aromatization was similar to that achieved by lesioning this locus. Thus, hippocampal aromatization, much of which is achieved at the synapse in this species, is critical for spatial memory performance.

Sex steroid levels and AD-like pathology in 3xTgAD mice.

Decreases in testosterone and 17β‐oestradiol (E2) are associated with an increased risk for Alzheimers disease (AD), which has been attributed to an increase in β‐amyloid and tau pathological lesions. Although recent studies have used transgenic animal models to test the effects of sex steroid manipulations on AD‐like pathology, almost none have systematically characterised the associations between AD lesions and sex steroid levels in the blood or brain in any mutant model. The present study evaluated age‐related changes in testosterone and E2 concentrations, as well as androgen receptor (AR) and oestrogen receptor (ER) α and β expression, in brain regions displaying AD pathology in intact male and female 3xTgAD and nontransgenic (ntg) mice. We report for the first time that circulating and brain testosterone levels significantly increase in male 3xTgAD mice with age, but without changes in AR‐immunoreactive (IR) cell number in the hippocampal CA1 or medial amygdala. The age‐related increase in hippocampal testosterone levels correlated positively with increases in the conformational tau isoform, Alz50. These data suggest that the over‐expression of human tau up‐regulate the hypothalamic‐pituitary‐gonadal axis in these mice. Although circulating and brain E2 levels remained stable with age in both male and female 3xTgAD and ntg mice, ER‐IR cell number in the hippocampus and medial amygdala decreased with age in female transgenic mice. Furthermore, E2 levels were significantly higher in the hippocampus than in serum, suggesting local production of E2. Although triple transgenic mice mimic AD‐like pathology, they do not fully replicate changes in human sex steroid levels, and may not be the best model for studying the effects of sex steroids on AD lesions.

Steroid profiling reveals widespread local regulation of glucocorticoid levels during mouse development.

Glucocorticoids (GCs) are produced by the adrenal glands and circulate in the blood to coordinate organismal physiology. In addition, different tissues may independently regulate their local GC levels via local GC synthesis. Here, we find that in the mouse, endogenous GCs show tissue-specific developmental patterns, rather than mirroring GCs in the blood. Using solid-phase extraction, HPLC, and specific immunoassays, we quantified endogenous steroids and found that in tissues of female and male mice, (1) local GC levels can be much higher than systemic GC levels, (2) local GCs follow age-related patterns different from those of systemic GCs, and (3) local GCs have identities different from those of systemic GCs. For example, whereas corticosterone is the predominant circulating adrenal GC in mice, high concentrations of cortisol were measured in neonatal thymus, bone marrow, and heart. The presence of cortisol was confirmed with liquid chromatography-tandem mass spectrometry. In addition, gene expression of steroidogenic enzymes was detected across multiple tissues, consistent with local GC production. Our results demonstrate that local GCs can differ from GCs in circulating blood. This finding suggests that steroids are widely used as local (paracrine or autocrine) signals, in addition to their classic role as systemic (endocrine) signals. Local GC regulation may even be the norm, rather than the exception, especially during development.

Rapid Effects of an Aggressive Interaction on Dehydroepiandrosterone, Testosterone and Oestradiol Levels in the Male Song Sparrow Brain: a Seasonal Comparison

Across vertebrates, aggression is robustly expressed during the breeding season when circulating testosterone is elevated, and testosterone activates aggression either directly or after aromatisation into 17β‐oestradiol (E2) in the brain. In some species, such as the song sparrow, aggressive behaviour is also expressed at high levels during the nonbreeding season, when circulating testosterone is non‐detectable. At this time, the androgen precursor dehydroepiandrosterone (DHEA) is metabolised within the brain into testosterone and/or E2 to promote aggression. In the present study, we used captive male song sparrows to test the hypothesis that an acute agonistic interaction during the nonbreeding season, but not during the breeding season, would alter steroid levels in the brain. Nonbreeding and breeding subjects were exposed to either a laboratory simulated territorial intrusion (L‐STI) or an empty cage for only 5 min. Immediately afterwards, the brain was rapidly collected and flash frozen. The Palkovits punch technique was used to microdissect specific brain regions implicated in aggressive behaviour. Solid phase extraction followed by radioimmunoassay was used to quantify DHEA, testosterone and E2 in punches. Overall, levels of DHEA, testosterone and E2 were higher in brain tissue than in plasma. Local testosterone and E2 levels in the preoptic area, anterior hypothalamus and nucleus taeniae of the amygdala were significantly higher in the breeding season than the nonbreeding season and were not affected by the L‐STI. Unexpectedly, subjects that were dominant in the L‐STI had lower levels of DHEA in the anterior hypothalamus and medial striatum in both seasons and lower levels of DHEA in the nucleus taeniae of the amygdala in the breeding season only. Taken together, these data suggest that local levels of DHEA in the brain are very rapidly modulated by social interactions in a context and region‐specific pattern.

Locally elevated cortisol in lymphoid organs of the developing zebra finch but not Japanese quail or chicken.

Glucocorticoids are important for production of functional lymphocytes and immunity. In altricial neonates, adrenal glands are unresponsive and local glucocorticoid synthesis in lymphoid organs may be necessary to support lymphocyte development. Precocial neonates, in contrast, have fully responsive adrenal glucocorticoid production, and lymphoid glucocorticoid synthesis may not be necessary. Here, we found that in altricial zebra finch hatchlings, lymphoid organs had dramatically elevated endogenous glucocorticoid (and precursor) levels compared to levels in circulating blood. Furthermore, while avian adrenals produce corticosterone, finch lymphoid organs had much higher levels of cortisol, an unexpected glucocorticoid in birds. In contrast, precocial Japanese quail and chicken offspring did not have locally elevated lymphoid glucocorticoid levels, nor did their lymphoid organs contain high proportions of cortisol. These results show that lymphoid glucocorticoids differ in identity, concentration, and possibly source, in hatchlings of three different bird species. Locally-regulated glucocorticoids might have species-specific roles in immune development.

Testosterone and Corticosterone in the Mesocorticolimbic System of Male Rats: Effects of Gonadectomy and Caloric Restriction

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.

Tyramide Signal Amplification Permits Immunohistochemical Analyses of Androgen Receptors in the Rat Prefrontal Cortex

Research on neural androgen receptors (ARs) has traditionally focused on brain regions that regulate reproductive and aggressive behaviors, such as the hypothalamus and amygdala. Although many cells in the prefrontal cortex (PFC) also express ARs, the number of ARs per cell appears to be much lower, and thus, AR immunostaining is often hard to detect and quantify in the PFC. Here, we demonstrate that biotin tyramide signal amplification (TSA) dramatically increases AR immunoreactivity in the rat brain, including critical regions of the PFC such as the medial PFC (mPFC) and orbitofrontal cortex (OFC). We show that TSA is useful for AR detection with both chromogenic and immunofluorescent immunohistochemistry. Double-labeling studies reveal that AR+ cells in the PFC and hippocampus are NeuN+ but not GFAP+ and thus primarily neuronal. Finally, in gonadally intact rats, more AR+ cells are present in the mPFC and OFC of males than of females. Future studies can use TSA to further examine AR immunoreactivity across ages, sexes, strains, and different procedures (e.g., fixation methods). In light of emerging evidence for the androgen regulation of executive function and working memory, these results may help understand the distribution and roles of ARs in the PFC.

Neuropeptide Y and orexin immunoreactivity in the sparrow brain coincide with seasonal changes in energy balance and steroids

The transition between the breeding and nonbreeding states is often marked by a shift in energy balance. Despite this well‐known shift in energy balance, little work has explored seasonal differences in the orexigenic neuropeptides that regulate food intake in wild animals. Here we tested the hypothesis that free‐living male song sparrows (Melospiza melodia) show seasonal changes in energetic state, circulating steroids, and both neuropeptide Y (NPY) and orexin (OX) immunoreactivity. Nonbreeding song sparrows had more fat and muscle, as well as a ketone and triglyceride profile suggesting a greater reliance on lipid reserves. Breeding birds had higher plasma androgens; however, nonbreeding birds did maintain androgen precursors in circulation. Nonbreeding birds had more NPY immunoreactivity, specifically in three brain regions: lateral septum, bed nucleus of the stria terminalis, and ventral tegmental area. Furthermore, nonbreeding birds had more OX immunoreactivity in multiple brain regions. Taken together, the data indicate that a natural shift in energy balance is associated with changes in NPY and OX in a region‐specific manner.

Rapid effects of 17β-estradiol on aggressive behavior in songbirds: Environmental and genetic influences

&NA; Contribution to Special Issue on Fast effects of steroids. 17&bgr;‐estradiol (E2) has numerous rapid effects on the brain and behavior. This review focuses on the rapid effects of E2 on aggression, an important social behavior, in songbirds. First, we highlight the contributions of studies on song sparrows, which reveal that seasonal changes in the environment profoundly influence the capacity of E2 to rapidly alter aggressive behavior. E2 administration to male song sparrows increases aggression within 20 min in the non‐breeding season, but not in the breeding season. Furthermore, E2 rapidly modulates several phosphoproteins in the song sparrow brain. In particular, E2 rapidly affects pCREB in the medial preoptic nucleus, in the non‐breeding season only. Second, we describe studies of the white‐throated sparrow, which reveal how a genetic polymorphism may influence the rapid effects of E2 on aggression. In this species, a chromosomal rearrangement that includes ESR1, which encodes estrogen receptor &agr; (ER&agr;), affects ER&agr; expression in the brain and the ability of E2 to rapidly promote aggression. Third, we summarize studies showing that aggressive interactions rapidly affect levels of E2 and other steroids, both in the blood and in specific brain regions, and the emerging potential for steroid profiling by liquid chromatography tandem mass spectrometry (LC‐MS/MS). Such studies of songbirds demonstrate the value of an ethologically informed approach, in order to reveal how steroids act rapidly on the brain to alter naturally‐occurring behavior.