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Keywords:

  • Egr-1;
  • norepinephrine;
  • serotonin;
  • white-throated sparrow;
  • ZENK

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

Behavioral responses to social stimuli often vary according to endocrine state. Our previous work has suggested that such changes in behavior may be due in part to hormone-dependent sensory processing. In the auditory forebrain of female white-throated sparrows, expression of the immediate early gene ZENK (egr-1) is higher in response to conspecific song than to a control sound only when plasma estradiol reaches breeding-typical levels. Estradiol also increases the number of detectable noradrenergic neurons in the locus coeruleus and the density of noradrenergic and serotonergic fibers innervating auditory areas. We hypothesize, therefore, that reproductive hormones alter auditory responses by acting on monoaminergic systems. This possibility has not been examined in males. Here, we treated non-breeding male white-throated sparrows with testosterone to mimic breeding-typical levels and then exposed them to conspecific male song or frequency-matched tones. We observed selective ZENK responses in the caudomedial nidopallium only in the testosterone-treated males. Responses in another auditory area, the caudomedial mesopallium, were selective regardless of hormone treatment. Testosterone treatment reduced serotonergic fiber density in the auditory forebrain, thalamus, and midbrain, and although it increased the number of noradrenergic neurons detected in the locus coeruleus, it reduced noradrenergic fiber density in the auditory midbrain. Thus, whereas we previously reported that estradiol enhances monoaminergic innervation of the auditory pathway in females, we show here that testosterone decreases it in males. Mechanisms underlying testosterone-dependent selectivity of the ZENK response may differ from estradiol-dependent ones.© 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 455–468, 2013


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

No environment is constant, and the relative importance of some types of sensory stimuli, particularly social signals, changes over time. As a consequence, the neural and behavioral responses to those stimuli must also change in order to remain context-appropriate. Recent evidence from fish, frogs, birds, and mammals suggests that one powerful modulator is endocrine state; hormones can alter sensory responses, affecting perception from the “bottom up” (reviewed by Maney, 2010; Maney and Pinaud, 2011). Despite the ubiquity of this phenomenon, the neural processes underlying it are not well understood.

Seasonally breeding songbirds are ideal models in which to explore these processes, first because the behavioral relevance of auditory courtship signals changes dramatically with endocrine state, and second because their auditory responses are well-studied. In female white-throated sparrows (Zonotrichia albicollis) with breeding-typical levels of plasma estradiol (E2), hearing conspecific male song elicits courtship displays and induces the expression of plasticity-associated genes such as ZENK (egr-1) in auditory areas (Maney et al., 2006; Sanford et al., 2010). Throughout the auditory pathway, the ZENK response is “selective”, meaning the response is higher to male song than to a control sound, only in females with breeding-typical plasma E2 levels (Maney et al., 2006; Maney and Pinaud, 2011). This finding strongly suggests that steroid-dependent modulatory input brings information on endocrine state into auditory areas.

We hypothesize that this modulatory input comes from monoaminergic systems. The monoamines dopamine (DA), norepinephrine (NE), and serotonin are widely understood to shape the response properties of sensory neurons by engaging context-dependent filters and inducing selectivity (reviewed by Hurley et al., 2004; Hurley and Hall, 2011). Serotonergic and noradrenergic projections to the auditory forebrain are thought to affect the processing of song (reviewed by Castelino and Schmidt, 2010). Noradrenergic denervation of the forebrain reduces behavioral and neural responses to song as well as behavioral and neural selectivity for sexually stimulating song (Appeltants et al., 2002; Lynch and Ball, 2008; Vyas et al., 2008; Poirier et al., 2011). Finally, Velho et al. (2012) recently showed that noradrenergic transmission in the auditory forebrain is necessary and sufficient for ZENK expression in that region. Monoamines are thus already known to modulate both behavioral and neural responses to song.

Because monoaminergic systems are highly sensitive to gonadal steroids in songbirds and other vertebrates (Kritzer and Kohama, 1998, 1999; Barclay and Harding, 1990), they are excellent candidates for mediating steroid-dependent plasticity of sensory processing. Our previous work has shown that in female white-throated sparrows, E2 treatment increases the selectivity of ZENK responses in catecholaminergic regions of the brainstem, the number of catecholaminergic neurons in those regions, and the density of catecholaminergic and serotonergic innervation of auditory areas (LeBlanc et al., 2007; Maney et al., 2008; Matragrano et al., 2011, 2012a). We have also shown that hearing male song rapidly induces catecholaminergic and serotonergic activity in the auditory forebrain of females (Matragrano et al., 2012a, 2012b). These results support the hypothesis that monoaminergic systems may participate in seasonal changes in auditory processing in females, for whom the behavioral relevance of song changes dramatically according to season. We have, however, never tested this model in males.

To a male white-throated sparrow, male song heard during the breeding season indicates defense (or intrusion) of a breeding territory. Free-living males are more likely to respond to song playback during the breeding season when plasma testosterone (T) is elevated (reviewed by Maney and Goodson, 2011). Thus, like females, males alter their behavioral responses to conspecific song according to season and endocrine state. Their neural responses may change as well. Auditory brainstem responses change seasonally in a variety of songbirds including Carolina chickadees (Poecile carolinensis), tufted titmice (Baeolophus bicolor), white-breasted nuthatches (Sitta carolinensis), and white-crowned sparrows (Z. leucophrys) (Lucas et al., 2002, 2007; Caras et al., 2010). Phillmore et al. (2011) showed that in male black-capped chickadees (P. atricapillus), ZENK expression in the auditory forebrain was higher in response to conspecific than to heterospecific song only when males were in breeding condition. There is thus evidence for seasonal regulation of auditory responses in several species and at several levels of the auditory pathway.

The anatomical and functional organization of central auditory pathways in songbirds largely resembles those found in other vertebrates, including mammals (Fig. 1; reviewed by Maney and Pinaud, 2011). Auditory input is transduced in the cochlea, ascends through brainstem areas analogous to mammalian cochlear nuclei, and arrives at the dorsal lateral mesencephalic nucleus (MLd), the avian homolog of the mammalian inferior colliculus (Karten, 1967). MLd neurons send direct projections to the thalamic nucleus Ovoidalis (Ov), the avian homolog of the ventral medial geniculate (Karten, 1967). Ov projects to a pronounced lobe in the forebrain that contains auditory areas. Inside this lobe, the caudomedial nidopallium (NCM) receives input from the thalamo-recipient Field L and is heavily interconnected with the caudomedial mesopallium (CMM). NCM and CMM are analogous to the supragranular layers of mammalian auditory cortex (Vates et al., 1996) or to mammalian auditory association cortex (Pinaud and Terleph, 2008; Tremere et al., 2009) and respond selectively to behaviorally relevant stimuli. For example, the factors that affect behavioral responses to song, such as attractiveness or familiarity, also affect the expression of ZENK (e.g., Gentner et al., 2001; Maney et al., 2003; Terpstra et al., 2006). Changes in the behavioral relevance of a signal are thus expected to be accompanied by changes in the ZENK response to that signal (Maney et al., 2006).

image

Figure 1. Parasagittal view of the auditory pathway in songbirds. The auditory nerve enters the brainstem and arrives at the cochlear nucleus (CN, also called nucleus magnocellularis) which projects to the auditory midbrain (the dorsal portion of the lateral mesencephalic nucleus, or MLd). MLd projects to the core region of the auditory thalamus, also called nucleus ovoidalis (Ov). The Ov core projects to the thalamorecipient region of the auditory forebrain, Field L, which then projects to the caudomedial nidopallium (NCM). NCM is reciprocally connected to the caudomedial mesopallium (CMM). NCM and CMM are thought to be analogous to the supragranular layers of mammalian auditory cortex (Vates et al., 1996) or to mammalian auditory association cortex (Pinaud and Terleph, 2008; Tremere et al., 2009).

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In the current study, we investigated whether systemic T manipulation alters the ZENK response in the auditory system of male white-throated sparrows and whether those effects might be mediated by monoaminergic innervation. In males of this species, breeding-typical behaviors such as territorial song and other aggressive vocalizations can be induced in non-breeding individuals by plasma T treatment (Maney et al., 2009). We treated male white-throated sparrows with T or vehicle and exposed them to either conspecific male song or behaviorally irrelevant tones. We then examined how T affected (1) ZENK responses to the sounds, (2) the densities of serotonergic and noradrenergic fibers innervating the auditory pathway, and (3) the number of detectable noradrenergic cells in the locus coeruleus (LoC).

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

Animals

All procedures in this study adhered to NIH standards and were approved by the Emory University Institutional Animal Care and Use Committee. Twenty-three male white-throated sparrows were collected in mist nets on the Emory campus during fall migration. We included equal numbers of the two plumage morphs, white and tan (see Maney, 2008), and balanced morph across treatment and stimulus conditions in the study. Sex and morph were determined by polymerase chain reaction (Griffiths et al., 1998; Michopoulos et al., 2007). The birds were housed at the Emory animal care facility in indoor walk-in flight cages and supplied with food and water ad libitum. Day length was held constant at 10:14 h light-dark, which corresponds to the shortest day the birds would experience during the winter at the capture site. The birds were kept under these conditions for at least 4 months to ensure that they were not photorefractory prior to the start of the study (Wolfson, 1958).

Hormonal Manipulation

All birds were transferred to individual cages (38 × 38 × 42 cm) inside sound-attenuated booths (Industrial Acoustics, Bronx, NY) before the start of the experiment. Day length remained at 10:14 h light–dark throughout the study. Each booth held four to six singly-housed males. On the day each bird was transferred, it received a subcutaneous silastic capsule (length 15 mm, ID 1.47 mm, OD 1.96 mm, Dow Corning, Midland, MI) sealed at both ends with A-100S Type A medical adhesive (Factor 2, Lakeside, AZ) and either left empty (n = 11) or filled with T (n = 12; Steraloids, Newport, RI). This dose of T increases plasma levels to breeding-typical levels in non-breeding males of this species (Maney et al., 2009). Hormone treatment was balanced within plumage morph and housing groups.

Sound Stimuli

The playback protocol has been previously described (Maney et al., 2006, 2009; Sanford et al., 2010). Briefly, recordings of singing male white-throated sparrows were downloaded from the Borror Laboratory of Bioacoustics birdsong database. They were then spliced together such that the song of a single male was heard every 15 s. In order to help overcome habituation to the stimulus, the singer's identity changed to a new male every 3 min. Each presentation contained the songs of 14 males, heard for 3 min each, in a unique order (total 42 min). In addition to the song presentations, we made tone presentations to control for exposure to an auditory stimulus. Fourteen unique tone sequences were made, each matched to one of the male songs with regard to duration, sound energy at each frequency, and number of onsets and offsets. The tone presentations were constructed exactly as were the song presentations, with tone sequences every 15 s and changing to a new sequence every 3 min, for 42 min.

Playback Experiment

The playback experiment began seven days after the onset of hormone treatment (Maney et al., 2009; Sanford et al., 2010). On the evening before stimulus presentation, each bird was isolated in an empty sound-attenuated booth equipped with a microphone, speaker, and video camera. Sound playback began approximately 1 h after lights-on the following morning. Half of each treatment group heard either songs (T-treated, n = 6; blank, n = 6) or tones (T-treated, n = 6; blank, n = 5), delivered via the speaker inside the booth at a peak level of 70 dB measured at the bird's cage.

Tissue Collection

Sixty min after the start of sound presentation, each bird was deeply anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL) and a blood sample was collected from the jugular vein into a heparinized StatSampler tube (Iris, Westwood, MA), which was later centrifuged and the plasma harvested and frozen at −20°C. Brains were harvested, and the rostralmost 3 to 4 mm trimmed away to allow the fixative to access the ventricles. The brains were fixed by immersion in 5% acrolein, cryoprotected in 30% sucrose, and frozen at −80°C until sectioning. Testes were inspected to confirm a regressed state (≤1mm in diameter).

Immunohistochemistry

Brains were cut into three series of 50 µm coronal sections using a freezing sliding microtome. We immunolabeled the three series for ZENK, serotonin transporter (SERT), or dopamine beta-hydroxylase (DBH) using standard immunohistochemistry (IHC) protocols (Maney et al., 2003, 2006; Matragrano et al., 2011, 2012a). The specificity of each of the primary and the secondary antibodies was tested by omitting it in test sections and observing a complete loss of specific staining. After incubation in primary antibody (see below), sections were subsequently incubated in a biotinylated secondary antibody against rabbit IgG (Vector, Burlingame, CA) diluted 1:250, followed by the ABC method (Vector, Burlingame, CA). ZENK and DBH immunolabeling was visualized with nickel-enhanced diaminobenzidine (Shu et al., 1988) and SERT immunolabeling with diaminobenzidine without nickel. Each series of brain sections was processed in three separate runs of IHC in which the treatment (T or blank) and sound stimulus (tones or songs) were balanced across runs. Following IHC, all of the sections were mounted onto gelatin-subbed microscope slides, dehydrated, and coverslipped in DPX (Sigma, St. Louis, MO).

Antisera

ZENK was labeled with egr-1 antisera (Santa Cruz Biotechnology Cat# sc189; Santa Cruz, CA) diluted 1:8,000. The specificity of this antibody has been validated previously in songbirds (Mello and Ribeiro, 1998) and specifically in white-throated sparrows (Saab et al., 2010). All labeling in brain sections was eliminated at antibody concentrations of 1:25,000 by preadsorption of the antibody with 50 µg/mL of the egr-1 peptide (Santa Cruz Biotechnology Cat# sc189-P; Saab et al., 2010).

SERT is considered a more stable marker of serotonergic fibers than serotonin because it is less likely to be rapidly metabolized (Nielsen et al., 2006). To label SERT, we used a rabbit polyclonal antibody (1:5,000) generated against a synthetic peptide sequence corresponding to amino acids (602–622) of rat SERT coupled to keyhole limpet hemocyanin (ImmunoStar Cat# 24330, Hudson, WI). We previously validated its specificity in white-throated sparrow brain sections (Matragrano et al., 2012a). All labeling in brain sections was eliminated at antibody concentrations of 1:25,000 by preadsorption of the antibody with 50 µg/mL of the SERT peptide (ImmunoStar Cat# 24332). One drawback of labeling SERT rather than serotonin itself is that the cell bodies in the dorsal raphe are not labeled without colchicine treatment (Yamamoto et al., 1998). Most commercially available serotonin antibodies are raised against serotonin conjugated to paraformaldehyde (Hoffman et al., 2008); because we used acrolein rather than paraformaldehyde fixation in this study, we could not use such antibodies and therefore could not quantify cell bodies in the dorsal raphe in this study.

To label DBH, we used a polyclonal antibody (1:16,000) generated in rabbit against DBH purified from bovine adrenal medulla (ImmunoStar Cat# 22806). According to the manufacturer, the antibody detects a triplet at approximately 72 to 74 kDa in a Western blot. The antibody labels a NE-like distribution of cells and fibers in a variety of birds (e.g., Bailhache and Balthazart, 1993; Karle et al., 1996; Castelino and Ball, 2005; Sockman and Salvante, 2008), including white-throated sparrows (LeBlanc et al., 2007). Sockman and Salvante (2008) reported that preadsorption with antigen supplied by the manufacturer of the antibody eliminates all labeling in brain sections from European starlings (Sturnus vulgaris).

Regions of Interest

We quantified immunoreactivity within NCM and CMM of the auditory forebrain, the auditory thalamus (Ov), the auditory midbrain (MLd), and the LoC. Images of all regions of interest were acquired using the 4× objective (NCM, MLd) or the 10× objective (CMM, Ov) on a Zeiss Axioskop attached to a Leica DFC480 camera and Macintosh G5 computer. All light and shutter speed settings were held constant for each photo. Images of each region were acquired from three or four consecutive sections (1 in 3 series) spanning 300 to 450 µm (see below). Images were approximately 46 MB in size and were converted to 8-bit with ImageJ (version 1.41o, National Institutes of Health, Bethesda, MD).

Caudomedial Mesopallium

CMM was photographed in four consecutive sections, spanning 450 µm, at the level where the lamina arcopallialis dorsalis meets the lateral ventricle (Maney et al., 2006; Maney and Pinaud, 2011) [Fig. 2(A)]. Using the freehand tool in ImageJ, the area of hippocampus in each image was subtracted before immunoreactivity was quantified.

image

Figure 2. Sound-induced expression of the protein product of ZENK, an immediate early gene. Photos depict typical responses in a testosterone (T)-treated bird hearing song. Immunoreactivity was quantified, within the areas shaded in the diagram, in (A) the caudomedial mesopallium (CMM), (B) the caudomedial nidopallium (NCM), (C) the shell and core of the auditory thalamus (Ov shell, Ov core), and (D) the auditory midbrain (MLd). ZENK induction was selective for song over frequency-matched tones in CMM regardless of hormone treatment. In NCM, the response was selective only in T-treated males. There was no effect of treatment on the ZENK responses in Ov or MLd. *p < 0.05 compared with the blank condition. See text for p values. Scale bar in (A), 200 µm, applies to all photos. Hp, hippocampus. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Caudomedial Nidopallium

NCM was photographed in four consecutive sections spanning 450 µm. The area sampled corresponded to the domain referred to previously as rdNCM (Sanford et al., 2010; Maney and Pinaud, 2011), which, in females, responds to conspecific song with robust upregulation of ZENK. ImageJ was used to select a circle with a diameter of 500 µm, approximately 350 µm from the medial edge of NCM (Maney and Pinaud, 2011) [Fig. 2(B)].

Nucleus Ovoidalis

Ov was photographed in three consecutive sections spanning 300 µm, with reference to Durand et al. (1992). Other investigators have reported that the core of Ov does not contain serotonin immunoreactivity (Zeng et al., 2007). In our material, we found likewise that the core contained minimal specific SERT-IR [Fig. 3(C)] and that the background was too high to select fibers accurately; therefore only the shell of Ov was sampled in sections immunolabeled for SERT. In the ZENK- and DBH-immunolabeled series, the boundary between the core and the shell was not clear. Most of the ZENK expression in Ov was located in a small area that spanned the medial boundary between the core and shell [see Fig. 2(C)] and we were thus not confident assigning the ZENK labeling to one compartment over another. We therefore combined both regions into one area for sampling ZENK- and DBH-immunoreactivity.

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Figure 3. Fibers immunoreactive for serotonin transporter (SERT; A–E) or dopamine β-hydroxylase (DBH; F–J) in the caudomedial mesopallium (CMM; A, F), caudomedial nidopallium (NCM; B, G), the auditory thalamus (Ov; C, H), auditory midbrain (MLd; D, I), and the apical hyperpallium (HA; E, J). SERT immunoreactivity was quantified in the Ov shell but not the core (C), because the core lacked specific labeling (see also Zeng et al., 2007). All images are from a blank-treated bird. Testosterone (T) treatment reduced SERT immunoreactivity in NCM, Ov, and MLd (K) and DBH immunoreactivity in MLd (L). Scale bar in (A), 50 μm, applies to all photos. *p < 0.05, see text for p values.

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Dorsal Portion of the Lateral Mesencephalic Nucleus

MLd was photographed in three (ZENK) or four (DBH and SERT) consecutive sections spanning 300 to 450 µm. The sections that contained the largest cross-sections of MLd were photographed and the borders of MLd traced with respect to the nucleus intercollicularis and surrounding tissue using the freehand tool of ImageJ (Maney et al., 2006) [Fig. 2(D)].

Apical Hyperpallium

In the DBH- and SERT-labeled sections, we photographed a non-auditory area, the apical hyperpallium (HA; Reiner et al., 2004), in the rostral-most section available for each brain (approximately A4.0 to A3.0, see Stokes et al., 1974). These photos were taken to assess whether the effects of T treatment might be general to sensory regions and not limited to auditory areas. The entire photo of HA (approximately 870 µm × 690 µm, taken with the 10× objective) was used to estimate fiber density.

Image Analysis

ZENK-immunoreactive (IR) cell nuclei and DBH- and SERT-IR fibers were selected in each image by a blind observer using the thresholding feature in ImageJ as previously described (Maney et al., 2003, 2006; LeBlanc et al., 2007; Matragrano et al., 2011, 2012a). Our method of thresholding has been validated and shown to have high interrater reliability and low variability (Matragrano et al., 2011). In most cases, we used the threshold set automatically by ImageJ, which is based on contrast. In rare cases, because of an artifact or higher than average background, the threshold chosen by the computer software was in obvious disagreement with actual labeled cells or fibers; in those cases the threshold was set manually. Accurate selection of cells and fibers was confirmed by the same observer for each label, with the same lighting and computer monitor. The area covered by ZENK-IR nuclei or by DBH-IR or SERT-IR fibers was then measured within each area of interest. To estimate the density of ZENK-IR nuclei and DBH- and SERT-IR fibers, we divided the total area selected, in square microns, by the total area sampled for each region in square mm. That value was then used in the statistical analysis (see below). Because ZENK-IR cells overlap in the images when expression is high, causing multiple cells to be counted by ImageJ as one object, we quantified the area covered to more accurately assess ZENK induction (Maney et al., 2006; Sanford et al., 2010).

Noradrenergic Neurons

DBH-IR neurons were counted using design-based stratified random sampling (Guena, 2000) throughout the extent of the LoC in the DBH-labeled series. Neurons on both sides of the brain were counted. Because SERT is not easily visible in cell bodies without colchicine treatment (Yamamoto et al., 1998; see above), we could not quantify serotonergic cells accurately in our material.

Radioimmunoassay

Plasma T levels were determined by the Biomarkers Core at Emory University using a commercially prepared kit produced by Diagnostic Systems Laboratories (Webster, TX); #DSL-4000. Samples were run in duplicate. This kit has been used to assay plasma T in a variety of avian species (Sandell, 2007; Casagrande et al., 2011; Müller et al., 2011; Pfannkuche et al., 2011). The cross-reactivity provided by the manufacturer was 5.8% with dihydrotestosterone, 2.3% with androstenedione, and <0.5% for other androgens. Sample volumes were 50 µL and the lower limit of detection was 0.05 ng/mL. Intra-assay variation was 6.3%. All samples were run in the same assay, eliminating interassay variation.

Statistical Analysis

All data were square root transformed to normalize their distribution. Because we collected data on multiple markers and brain regions in the same set of animals, we could not assume these variables were independent. We therefore began our analysis with a repeated-measures MANOVA with region of interest (NCM, CMM, Ov, or MLd) and marker (ZENK, SERT, or DBH) as the within-subject factors, and hormone treatment (blank or T) as a between-subjects factor. Because the two plumage morphs differ with respect to their singing behavior (Falls and Kopachena, 2010) and plasma levels of T during the breeding season (Spinney et al., 2006), we also included morph as a between-subjects factor. Data from LoC were excluded from this test because we did not have data on all three markers in that region. We also excluded from this test one bird for which we had data only on MLd and LoC. Following this initial omnibus test, we performed univariate F-tests to look for effects in each region of interest for each marker as outlined below.

ZENK

For ZENK, we first performed MANOVAs with IHC run (1st, 2nd, or 3rd group of sectioned brains labeled) to rule out main effects and interactions with each other factor (treatment, stimulus, and morph) independently. We then ran univariate F-tests to test for effects of treatment, stimulus, and morph on ZENK immunoreactivity in each region of interest. When significant effects were found, we performed univariate F-tests to look for effects of treatment within stimulus and effects of stimulus within treatment, and interactions between both of those factors and morph.

DBH and SERT

We first used ANOVAs with stimulus and IHC run as between-subjects factors to confirm that there were no effects of stimulus on DBH-IR or SERT-IR in any region. We then performed univariate F-tests for each marker in each region of interest with hormone treatment, morph, and IHC run as fixed factors. When significant effects were found, we performed univariate F-tests to look for effects of treatment and morph.

Digital Photography

To make the figures, the brightness of photomicrographs was adjusted so that the background in images within a single figure was comparable.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

The T implants brought plasma T levels up to 9.72 ± 0.87 ng/mL (range 5.44–14.48 ng/mL) which is in the range reported for free-living territorial males (Spinney et al., 2006; Swett and Breuner, 2008). Using a different T assay (as per Stevenson et al., 2012), we have independently confirmed plasma T levels in this range in free-living males early in the breeding season, when males are setting up territories (Horton, unpublished data). The level we achieved with the implants in this study is quite typical of a free-living white-striped male during the prelaying stage (mean 10.93 ± 0.99 ng/mL, range 5.42–20.32 ng/mL, n = 18; Horton, unpublished data). Plasma T in blank-implanted males remained at non-breeding levels (0.462 ± 0.15 ng/mL). There was a highly significant effect of T-treatment (ANOVA: F(1,21) = 160.564, p < 0.001). Plasma T did not differ between the morphs (F(1,21) = 0.299, p = 0.591), and there was no interaction between hormone treatment and morph (F(1,21) = 0.418, p = 0.526).

When we analyzed the data on all of the markers and brain regions in a repeated-measures MANOVA (see Methods), we found a main effect of hormone treatment (F(1,18) = 6.428; p = 0.021). There was no main effect of morph (F(1,18) = 1.564; p = 0.227) but there was an interaction between morph and treatment (F(1,18) = 4.578; p = 0.046). We then proceeded to analyze the data for each marker separately (see below). Because we considered morph to be a nuisance variable rather than a factor of high interest, we present any further effects or interactions in a separate paragraph below, at the end of the Results section.

ZENK Immunoreactivity in Auditory Areas

The ZENK responses to song and tones are graphed in Figure 2. Post hoc univariate F-tests with T treatment and sound stimulus as fixed factors showed an effect of stimulus in NCM (F(1,21) = 8.668, p = 0.011) and CMM (F(1,21) = 16.566, p = 0.001). In CMM, hearing song induced more ZENK-IR than did tones regardless of hormone treatment [T: F(1,11) = 7.728, p = 0.024; blank: F(1,11) = 12.427, p = 0.012; Fig. 2(A)]. In NCM, there was an interaction between stimulus and treatment (F(1,21) = 5.182, p = 0.039); in T-treated males, the ZENK response to song was significantly greater than to tones [F(1,11)= 12.071, p = 0.008; Fig. 2(B)] whereas in blank-treated males, the responses to song and tones were indistinguishable (F(1,9) = 0.310, p = 0.598). There were no effects of stimulus or treatment on ZENK-IR, nor any interactions between the two, in the auditory thalamus or midbrain. Although T treatment increases song rate, particularly in response to playback (Maney et al., 2009), we have previously shown that ZENK expression in our regions of interest is unlikely to be affected by the birds' own vocalizations (Sanford et al., 2010).

SERT Immunoreactivity in Auditory Areas

The effect of T treatment on SERT immunoreactivity is plotted in Figure 3(K). T-treatment significantly reduced the density of SERT-IR fibers in NCM (F(1,21) = 8.355, p = 0.015), the shell of Ov (F(1,21) = 11.478, p = 0.006), and MLd (F(1,22) = 20.886, p = 0.001) but not CMM. There was no effect of treatment in the visual area HA.

DBH Immunoreactivity in the LoC and Auditory Areas

The effect of T treatment on DBH immunoreactivity is plotted in Figures 3(L) and 4. T treatment significantly increased the number of DBH-IR cells in the LoC (F(1,22) = 5.053, p = 0.046), but decreased DBH-IR fiber density in the auditory midbrain (MLd) (F(1,22) = 6.708, p = 0.025). There were no main effects of treatment in any other region of interest, There was no effect of treatment in the visual area HA.

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Figure 4.  Effects of testosterone (T) treatment on noradrenergic cells. (A) Cell bodies immunoreactive (IR) for dopamine beta-hydroxylase (DBH) in the locus coeruleus. Scale bar, 100 µm. (B) T-treated birds had more DBH-IR cells than those treated with blank capsules. *p < 0.05. Scale bar, 100 µm.

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Effects of Plumage Morph

There was a main effect of morph only on ZENK expression in MLd (F(1,21) = 4.664, p = 0.049) but it did not interact with treatment or stimulus in that region. We noted some interesting interactions between morph and treatment in the other regions, however. First, the effect of T-treatment on SERT-IR in the auditory forebrain depended on morph. In both CMM and NCM, morph interacted with treatment (CMM, F(1,21) = 9.934, p = 0.009; NCM, F(1,21) = 9.761, p = 0.010) such that T-treatment reduced SERT-IR fiber density in white birds (CMM, F(1,21) = 1.220, p = 0.306; F(1,21) = 11.994, p = 0.005; NCM, F(1,21) = 12.101, p = 0.005) but not tan birds (CMM, NCM, F(1,8) = 0.117, p = 0.742). We also found a three-way interaction between the effects of stimulus, treatment, and morph in NCM (F(1,21) = 6.952, p = 0.020), but could not analyze that interaction further because of low sample sizes within the three variables of morph, stimulus, and treatment. Finally, we found an effect of T-treatment on DBH-IR in Ov that depended on morph (F(1,21) = 5.364, p = 0.041) such that T treatment decreased DBH fiber density in the tan (F(4,8) = 21.714, p = 0.010) but not the white birds (F(5,12) = 0.481, p = 0.510). Follow-up studies will be needed to explore further the hypothesis that T has different effects in the two morphs.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

We have hypothesized that gonadal steroids alter the salience of reproductively relevant signals, facilitating responses during the breeding season. We previously showed that in female white-throated sparrows, sound-induced ZENK responses in NCM were selective for conspecific songs over control sounds only when plasma E2 reached breeding-typical levels (reviewed by Maney and Pinaud, 2011). We show here that in males, increasing T to breeding-typical levels could induce that selectivity as well. NCM may therefore respond more selectively to conspecific song during the breeding season in both males and females.

The effect of T on selectivity in NCM was not attributable to T-induced increases in the response to song. Hearing song resulted in similar ZENK induction in both T-treated and placebo-treated birds [Fig. 2(B)]. Rather, T may have affected selectivity by reducing the ZENK response to tones. In black-capped chickadees, the ZENK response to heterospecific song was strikingly lower throughout the auditory forebrain in breeding males than in non-breeding males (Phillmore et al., 2011). Our results show that this effect was likely attributable to seasonal changes in reproductive hormones. In female white-throated sparrows, E2 treatment inhibited the ZENK response to non-relevant synthetic tones (Maney et al., 2006). Overall, our results point to steroid-dependent modulatory inputs that promote selective responses to behaviorally relevant stimuli by inhibiting responses to non-relevant stimuli. In young zebra finches learning song, ZENK expression in NCM is constitutively high, even during silence, and the development of a selective ZENK response in NCM coincides with a rise in T levels (Hutchison et al., 1984; Stripling et al., 2001). We have shown that in a seasonally breeding species, selectivity may be induced in both sexes by hormone treatment that mimics adult breeding levels.

Although the effects of T and E2 on the selectivity of the ZENK response are similar in direction, these steroids seem to have opposite effects on monoaminergic innervation of the auditory pathway. Whereas E2 treatment of non-breeding females increased the density of SERT-IR and DBH-IR fibers in the auditory forebrain and midbrain (Matragrano et al., 2011, 2012a), T treatment of males decreased it in many of the same regions [Fig. 3(K,L)]. Thus, if hormone-dependent changes in auditory selectivity are mediated by monoaminergic activity, T and E2 likely act via different monoaminergic mechanisms. Finally, whereas E2 treatment increased the number of DBH-IR cells in the LoC in females, T treatment reduced that number in males (Fig. 4). Initially it may seem paradoxical that T increased the number of detectable cell bodies while reducing fiber density. This result may indicate decreased transport of DBH to the terminals from the cell bodies. Alternatively, it may suggest either increased release or increased retrograde transport of inactive enzyme, both of which may stimulate increased synthesis in the cell bodies (Axelrod, 1972).

E2 has been reported to enhance monoaminergic activity (Sumner and Fink, 1998; McQueen et al., 1999; Cornil et al., 2006; Matragrano et al., 2011, 2012a, 2012b; cf. Kabelik et al., 2011). In contrast, androgens seem to have an inhibitory effect. In rodents, castration increases and androgen treatment reduces monoamine content, turnover, or fiber density (Battaner et al., 1987; Gabriel et al., 1988; Bitar et al, 1991; Kritzer, 2003; Grimes et al., 2006; c.f. Bradshaw et al., 1982). Several studies in which steroids were not directly manipulated suggest a negative relationship between plasma androgens and monoamine activity (Juorio et al., 1991; Blanchard et al., 1993; Easterbrook et al., 2007). Negative relationships have also been reported in fish (Elofsson et al., 2000; Hernandez-Routa et al., 2002), indicating that the phenomenon may be widespread in vertebrates. In rodents, when monoamine activity was reduced by castration, it was restored by treatment with E2 or T but not dihydrotestosterone (DHT) (Sumner and Fink, 1998; McQueen et al., 1999), which suggests that enhancing effects of T occur via aromatization and action on ER. In birds, androgens could act directly on the LoC, ventral tegmental area (VTA), substantia nigra (SN) and central gray (GCt), which contain androgen receptors (AR; Balthazart et al., 1998; Maney et al, 2001). T converted to E2 could act via estrogen receptors (ER) in the LoC and VTA (Maney et al., 2001).

We hypothesize that the effects of sex steroids on monoaminergic systems may, in part, explain hormone-induced ZENK selectivity in auditory areas. We showed previously in females (reviewed by Maney and Pinaud, 2011) and now in males that sex steroids altered both the monoaminergic fiber density and the selectivity of ZENK responses in at least some of those areas. We have not, however, shown evidence of a causal relationship between monoamines and ZENK. Velho et al. (2012) found that in zebra finches, application of NE directly to NCM induced a robust ZENK response. Further, the ZENK response to song was prevented by noradrenergic receptor blockade. In the present study, however, we have shown only a correlational relationship between monoaminergic innervation and ZENK selectivity. We should therefore consider the alternative—but not mutually exclusive—possibility that sex steroids act directly on steroid receptors in auditory areas, independently of monoamines.

There is little or no AR in the auditory pathway of songbirds (Balthazart et al., 1992; Bernard et al., 1999; Metzdorf et al., 1999; Gahr, 2001). Any direct effects of T on ZENK induction must therefore occur primarily via aromatization and action on ER. In this study the effects of T on ZENK expression were limited to NCM, which is the only region of the auditory pathway that expresses ER or aromatase (ARO) (Gahr et al., 1993; Balthazart et al., 1996; Bernard et al., 1999; Metzdorf et al., 1999; Gahr, 2001). Thus, it is possible that the effects of T on ZENK selectivity observed in this study may be explained without invoking an indirect mechanism. When considering our results from females, however, it becomes clear that E2 affects ZENK responses indirectly. In females, E2 treatment induces selectivity not only in NCM but also in CMM, Ov, and MLd (Maney et al., 2006; Maney and Pinaud, 2011). Because only NCM contains ER, E2-dependent ZENK selectivity in other auditory regions cannot be explained by direct effects of E2. Extrinsic or descending modulatory inputs must be involved. Because of the widespread effects of sex steroids on monoaminergic fiber density, and the clear role of monoaminergic transmission on the auditory ZENK response (Velho et al., 2012), these neuromodulators represent excellent candidates.

The extensive steroid-induced selectivity in females, compared with the rather limited induction of such in males, implies a sex difference in the mechanisms underlying auditory ZENK responses. Such a difference would be consistent with Hoke et al. (2010), who reported that in túngara frogs (Engystomops pustulosus) listening to male calls, midbrain ZENK responses predicted forebrain responses in females but not males. Their result suggested a sexual dimorphism in sensory-motor gating between auditory inputs and forebrain integration centers. Any sex comparisons from our work, however, are confounded by the fact that we administered E2 to females and T to males. The males in this study likely had limited ability to convert exogenous T to E2. Although ARO activity is present during the winter in seasonally breeding sparrows (Schlinger et al., 1992), it is much lower than in the spring (Soma et al., 2003). Photostimulation, in addition to T-treatment, is necessary to induce spring-like ARO activity (Meitzen et al., 2007). We have found that although plasma E2 levels do not differ between breeding males and females in a free-living population of white-throated sparrows (Horton, unpublished data); administration of T to captive males housed on short days does not significantly elevate plasma E2 (Grozhik, unpublished data). Because the birds in this study were not photostimulated and presumably had low ARO activity, exogenous T could act via ER only in brain regions that contain sufficient active ARO. Those regions include NCM, where we saw an increase in ZENK selectivity, but not other auditory regions or monoaminergic cell groups (see Balthazart et al., 1996). Manipulations of T, DHT, and E2 will be necessary to understand more fully the regulation of ZENK selectivity and monoaminergic innervation of the auditory pathway in males.

White-throated sparrows occur in two plumage morphs, white and tan, that differ with respect to aggression, parental behaviors, and plasma T (reviewed by Maney, 2008). When plasma T is experimentally equalized between the morphs, behavioral differences persist (Maney et al., 2009), suggesting that the morphs may be differentially sensitive to circulating gonadal steroids. The results of the current study are consistent with this hypothesis. T treatment reduced the density of SERT-IR fibers in NCM in white birds only, and DBH-IR fibers in Ov in tan birds only. Because we had only two to four birds of each morph within each of our four treatment-stimulus groups, we were unable to fully explore whether the effect of T treatment on the ZENK response in NCM depended on morph. Here we can report only a significant interaction between morph, stimulus, and treatment, which is consistent with that hypothesis. The plumage coloration segregates absolutely with a large rearrangement of chromosome 2 (Thorneycroft, 1975; Thomas et al., 2008), which contains neuroendocrine genes, such as ER-alpha and 5-alpha reductase (see Thomas et al., 2008), that contribute to aggression and parental behavior. Variation in the expression of steroid-sensitive genes contributes to variation in aggressive behavior (Rosvall et al., 2012). We are currently characterizing the neural expression of these genes in both morphs.

Acknowledgments

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES

The authors thank Chris Goode, Joanna Hubbard, Susie Lackey, Henry Lange, Vasiliki Michopoulos, Arundhati Murthy, Sara Sanford, and Jim Thomas for technical assistance, and the Department of Biology at Emory University for providing resources.

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  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES
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