All procedures were approved by the UCLA institutional animal care and use committee. All brain tissue and plasma were collected from captive adult zebra finches. Within <2 min of disturbance (to lessen effects of stress on steroid levels) birds were rapidly decapitated and the whole brain was removed, dissected into brain macroareas (see below), and stored at −80°C until homogenization. Blood was collected from the trunk and centrifuged at 10,000 rpm for 2 min. The resulting plasma was stored at −80°C until extraction.

We initially tested the ability of the commercial assay to detect E₂ in brain homogenates using conventional liquid (organic) extraction methods with diethyl ether. Adult female zebra finches were isolated for 24 h prior to estradiol or saline injection into the pectoralis muscle. Estradiol treated birds (n = 3) were given 20 μl of 300 μg/ml E₂ solution in saline (Sigma). Saline treated birds (n = 3) were given 20 μl of saline (0.9% w/vol). Injections were administered 1 h prior to tissue collection, whereupon the brain was collected and immediately dissected onto iced aluminum foil into functional regions using established landmarks (Remage-Healey et al., 2009): anterior telencephalon (AT), posterior telencephalon (PT), hippocampus (HP), mediobasal telencephalon, which contains aromatase-rich nucleus taeniae (MBT), anterior hypothalamus (AH), posterior hypothalamus (PH), optic tectum (OT), and cerebellum (CB). Brain tissues were weighed immediately after dissection and frozen at −80°C until further processing.

Prior to assaying, samples were thawed, homogenized in SPO₄ buffer on ice and ether extracted (see below). Samples were then reconstituted in 120 μl of EIA buffer and assayed on an E₂ EIA kit (Cayman Chemical) for measurements of E₂ levels.

Whole homogenates were prepared in 2 ml of SPO₄ buffer on ice via a homogenizer (Tissue Tearor, Biospec) until complete dispersion of tissue into the buffer. Each sample was then ether extracted by first adding 3 ml of diethyl ether, vortexing for 30 s, and centrifuging at 2100 rpm for 5 min at 40°C. The resulting mixture was then incubated in a MeOH/dry ice bath to solidify the aqueous phase (bottom) and the organic phase was eluted into a new tube. Ether extraction was repeated twice more on the thawed aqueous phase of each sample. Following elution, the organic phase was dried under air in a water bath at 50°C prior to being resuspended in 120 μl EIA buffer for either EIA directly or SPE.

We utilized a 12-port Visiprep™ SPE vacuum manifold (Supelco) with endcapped C18-SD 3 ml cartridges (Empore). Initial experiments showed that extracting homogenized brain tissue with SPE alone (i.e., without prior organic extraction) severely curtailed recovery of E₂ to as much as 10% (preliminary data, not shown), due to homogenized tissue obstructing the flow of eluent through the vacuum manifold. Therefore, we performed a two-stage extraction (ether extraction as above, followed by SPE) on brain tissue and plasma samples to determine the effectiveness of this dual extraction method at reducing EIA assay interference.

Prior to eluting samples, C18 columns were conditioned with 250 μl of 100% MeOH and equilibrated twice with 250 μl ddH₂O under vacuum pressure. Dried ether extracted samples were resuspended in 120 μl of EIA buffer, loaded, and eluted through the columns under vacuum pressure. After this initial elution, columns were eluted with 250 μl of ddH₂O twice to wash out hydrophilic polar compounds. A third and fourth wash with 250 μl of 90% MeOH then allowed for the elution of relatively hydrophobic compounds, including steroids (e.g., Newman et al., 2008). Columns were dried under vacuum after each loading. For each sample, eluent derived from the 90% MeOH elution was evaporated to dryness under a steady stream of air in a water bath at 50°C and stored at −20°C. Prior experiments using gas chromatography (GC)/mass spectrometry (MS) confirmed that the 90% MeOH fraction fully eluted estradiol (Remage-Healey and Schlinger, unpublished observations). Samples were resuspended with 120 μl of EIA buffer immediately prior to assaying.

Previously, we optimized the usage of the E₂ EIA (Cayman Chemicals) for the quantification of E₂ levels in microdialysate samples, validated with independent confirmation of E₂ using GC/MS; Remage-Healey et al., 2008). Here we assess the ability of EIA to accurately measure a gradient of exogenous E₂ concentrations in whole brain homogenate following two extraction protocols: extraction with ether only and extraction with ether followed by SPE.

Homogenized adult zebra finch brain tissue in SPO₄ buffer (pH – 7.4–6, 159.7 mg/ml) was separated into eight 500 μl aliquots. Each aliquot was spiked with radioinert E₂ to obtain one of eight concentrations to approximate the physiological range of E₂ levels in zebra finch tissue (4000, 1600, 640, 256, 102, 41, 16, 7 pg/ml) and in the dynamic range of the commercial EIA. Samples were ether extracted and then reconstituted in 250 μl of EIA buffer. Each sample was then split into two equal 120 μl aliquots to form two sets of eight 120 μl aliquots, and SPE was conducted on one set (as described above).

Ether and ether +SPE extracted samples were assayed on E₂ EIA along with unextracted standards of equivalent E₂ concentration (4000, 1600, 640, 256, 102, 41, 16, 7 pg/ml) to determine the effectiveness of the two extraction protocols in reducing assay interference for the displacement curve. We estimated the recovery of E₂ after ether extraction alone vs. ether extraction plus SPE. Recovery was estimated in two ways: (1) the extraction recovery of radiolabeled E₂ as determined via radioisotopic decay using a liquid scintillation counter and (2) the extraction recovery of radioinert E₂ as determined via EIA. We added 10,000 cpm of tritium-labeled E₂ (48.9 nM, [³H]-Estradiol; New England Nuclear) into a set of 2 ml SPO₄ aliquots in duplicate. Both sets were ether extracted and one set was resuspended with 120 μl of EIA buffer prior to SPE. Extracted aliquots were then reconstituted with 100 μl of methanol and vortexed for scintillation counting alongside unextracted but equivalently spiked samples. Radioinert E₂ was added to a separate set of 2 ml SPO₄ aliquots in quadruplicate. Both sets were ether extracted and one set was resuspended with 120 μl of EIA buffer prior to SPE. Extracted aliquots were then reconstituted with 2 ml of SPO₄ buffer and assayed via EIA alongside unextracted but similarly spiked samples. A similar extraction recovery was estimated for homogenized brain tissue in a separate experiment.

Using the optimized combined liquid and SPE procedure we measured differences in E₂ levels of dissected macroareas of the adult male zebra finch brain. Adult males (n = 12) were placed in sound isolation chambers 24 h prior to exposure to a stimulus consisting of two females placed in an adjacent cage inside the chamber. Immediately after 30 min of exposure to females, males were decapitated and whole blood was collected, centrifuged, and plasma was stored at −80°C. Brains were dissected on ice into functional groups, according to established procedures for isolating macroareas based on primary landmarks (see above; Remage-Healey et al., 2009). Immediately after dissection, tissue, and plasma wet weight was recorded and frozen at −80°C. Prior to assay, tissues were thawed, homogenized in SPO₄ buffer on ice, ether extracted, and solid phase extracted (as above). Finally, samples were reconstituted in 120 μl of EIA buffer and assayed on an E₂ EIA kit.

Compared to saline treated birds, we observed a clear elevation in overall E₂ levels across ether extracted brains of E₂ treated birds (df = 1,28 F = 30.686, p < 0.0001; Figure 1). Mann–Whitney post hoc tests revealed that E₂ levels were significantly elevated following E₂-injection in HP, AH, OT, nucleus taeniae, and PT (Z > 1.96; p < 0.05 for all comparisons) but not in AT or CB (p > 0.13). As expected, substantial variation in E₂ levels occurs across brain regions regardless of treatment (df = 6,28 F = 8.899, p < 0.0001). Therefore, the commercial EIA demonstrated that endogenous and exogenous E₂ was detectable in songbird brain tissue following ether extraction alone.

Recovery of estrogens following our extraction procedures was estimated using both radiolabeled and radioinert estrogens in separate extraction experiments. In SPO₄ solution, we observed a 93.4 and 86.2% recovery of radiolabeled and radioinert E₂, respectively, due to ether extraction alone. With ether extraction followed by SPE, we observed a total net recovery of 76 and 89.5% of radiolabeled and radioinert E₂, respectively (Figure 3). In a separate experiment, the total recovery of E₂ following ether +SPE extractions (as measured by EIA) from brain homogenate was 60.09 ± 4.02% (in quadruplicate). The results presented below for extracted samples does not account for recovery.

We compared the slopes of displacement curves that were extracted with ether alone vs. ether +SPE (Figure 2). The slope of the ether extracted dilution curve was indistinguishable from zero (F = −3.946, p = 0.121), and therefore is a poor predictor of brain E₂ content, particularly at lower levels. A multiple linear regression model indicated that the ether extracted curve was indeed not parallel to the standard curve (t = 9.708, p = 1.41e-08; Figure 2: box data labels). Our results strongly suggest that ether alone yields a particular poor extraction.

Enhancing extraction by combining ether followed by SPE, demonstrated a significant improvement in the slope of the displacement curve compared to the slope of the ether only extraction (t = 4.356, p = 0.0038). Taken together, the displacement curve extracted with ether +SPE demonstrated a significant improvement in dynamic sensitivity as compared to the displacement curve extracted with ether alone. Despite this improvement, the use of ether +C18 columns for the extraction of serially diluted E₂ spiked brain homogenate yielded a curve that was not strictly parallel with the standard curve (t = 5.352, p = 4.36e-05; Figure 2: triangle data labels). Importantly, however, the commercial EIA was better able to discriminate between differences in E₂ levels at lower concentrations (<650 pg/ml) with the addition of the SPE. This is particularly important since E₂ concentrations in zebra finch plasma and brain predominantly occur within this critical range.

Using our optimized ether +SPE protocol, EIA was used to measure brain E₂ levels in adult male zebra finches following 30 min of exposure to adult females (n = 12; Figure 4). There were significant regional differences in E₂ levels (df = 8,90 F = 3.025, p = 0.0047). MBT levels were highest, and were significantly greater than those found in telencephalon (Fisher’s PLSD: vs. AT: p = 0.0211, vs. PT: p = 0.028) and hypothalamus (vs. AH: p = 0.0081, vs. PH: p = 0.0057). MBT (containing aromatase-rich nucleus taeniae) and the HP both had E₂ levels significantly higher than plasma (MBT: p = 0.0011, HP: p = 0.0499), OT (MBT: p = 0.0003, HP: p = 0.0178), and CB (MBT: p = 0.0002, HP: p = 0.0152).

Our results show that the commercial E₂ EIA assay used here is capable of detecting exogenous increases in brain E₂ concentration. Further experiments showed that EIA is capable of detecting changes in ether extracted brain E₂ content induced by the potent aromatase inhibitor Fadrozole (35–37% decrease in brain E₂ content in telencephalon and HP; data not shown) and that ether extracted brain estrogens are detectable and unequivocally confirmed using GC/MS (see Results). These initial experiments confirmed the capacity of the commercial EIA to detect brain E₂ levels, and changes in brain E₂ content induced via endogenous and exogenous treatments. Importantly, however, the experiment with exogenous E₂-injection (Figure 1) alerted us to the potential for inaccuracies in steroid measurements at biological relevant levels using liquid extraction alone.

We tested the possibility that a two-stage extraction procedure was required to eliminate interfering substances from the EIA reaction plate. SPE, particularly reverse-phase extraction using C18 coated silica, has gained popularity for a variety of applications as a method of purifying samples for steroid analysis (see Introduction). In particular, SPE has been useful in optimizing the detection and quantification of brain E₂ content as measured by RIA (Newman et al., 2008). Here we provide an independent confirmation of the validity of this approach for brain E₂ content, and we demonstrate SPE’s potential to improve the performance of the EIA for the quantification of brain E₂, particularly at the low, subnanomolar concentrations.

Extraction prior to assaying plays an integral part in minimizing possible interference. Indeed, we observe that a limited liquid–liquid extraction of brain homogenate hinders EIA’s ability to detect variation of sample E₂ levels at the lower range of steroid concentrations, effectively “flattening” the displacement curve and rendering any measurement below approximately 600 pg/ml unreliable (Figure 2). Subsequent elution of samples through SPE greatly increased the assay’s sensitivity at the lower end of the curve in comparison to a liquid–liquid only extraction.

The most likely explanation for the improvements seen here with SPE is the elimination of substances that interfere with the EIA’s enzyme reaction(s). Assay interference can originate from a number of sources and can occur at different stages of the immunoassay. EIAs rely on the highly specific association between the primary antibody and the target antigen. In the case of competitive EIAs, the resulting antibody–antigen complex must also bind with high affinity to a plate-bound antibody IgG. The degree to which the extraction step minimizes cross-reactivity and non-specific binding during plate development likely directly impacts the accuracy and precision of the resulting colorimetric reaction step.

A number of substances may disrupt proper ligand–antibody competition, which can lead to the distortion of the displacement curve. While highly sensitive, the E₂-antibody used here has cross-reactivity for estradiol-3-glucuronide (14%), estrone (12%), and estradiol-17-glucuronide (10%). These and estrogenic derivatives and conjugates, such as E₂ fatty acid esters, have similar chemical properties to estradiol, and may remain in significant quantities after a simple ether extraction. Although little is known of the presence of these E₂ metabolites in birds, there is evidence that E₂ fatty acid esters are synthesized in the rat brain (Xu et al., 2002) and exert estrogenic effects on a variety of rat brain tissue (MacLusky et al., 1989). The presence of these conjugates may inflate the measurement of unconjugated E₂ levels by directly competing with free E₂ for antibody binding sites. In addition, E₂ fatty acid esters are known to associate with lipoproteins during circulation (Larner et al., 1987), granting them both lipophilic and hydrophilic properties that further complicate liquid extraction. These and other substances may persist in the saponified layer between the aqueous and organic phase during liquid (organic) extraction alone, and subsequently provide a source of interference during EIA. Residual non-estrogenic lipids, likely composed of free fatty acids and glycolipids, can prevent binding indirectly, by sequestering steroids or their antibodies within micelles (Rash et al., 1979). The forebrain of the zebra finch also contains robust expression of estrogen receptors (ERs; Gahr and Metzdorf, 1999) and unextracted ER and brain-derived E₂ binding proteins may all compete with the assay’s E₂-antibody for both free and conjugated E₂. The current findings are consistent with the hypothesis that the two-stage liquid/SPE protocol improves the separation of E₂ from such sources of assay interference.

Despite it’s safety and cost advantages, EIA may in fact be more sensitive to interfering substances than RIA. The quantification of bound antigen using EIA is achieved by first washing away free reagents, and then measuring the rate of a photochemical reaction (e.g., hydrolysis of acetylcholine by acetylcholinesterase) with a spectrophotometer. This process is susceptible to the presence of non-specific proteases that may significantly reduce the reactive properties of the EIA.

Using this optimized two-stage liquid/SPE protocol, the current results demonstrate that regional differences exist in brain E₂ content. The addition of SPE yielded results that are consistent with known patterns of aromatase activity and ER expression in the songbird brain (Saldanha et al., 2000). Areas with established aromatase expression showed higher concentration of E₂ in males, including mediobasal telencephalon containing nucleus taeniae, hypothalamus, HP, and telencephalon, in contrast to the CB and OT, which contain little to no known aromatase in the uninjured brain (Saldanha et al., 2000). The region-dependent ER expression throughout the zebra finch brain (Metzdorf et al., 1999) is also very likely to contribute to region differences in brain estrogen content as measured using these and similar methods, due to local accumulation and sequestration of neurally and peripherally derived estrogens. These findings for the region specificity of brain estrogen content, and the correspondence to brain aromatase and ER expression are presented in Figure 4. Lastly, the relative paucity of E₂ in plasma compared to brain regions such as the MBT and HP is consistent with the hypothesis that the brain is the primary source of estrogens in the male songbird (Schlinger and Arnold, 1991; Taves et al., 2010).