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. Author manuscript; available in PMC: 2012 May 19.
Published in final edited form as: Neuroscience. 2011 Mar 21;182:133–143. doi: 10.1016/j.neuroscience.2011.03.012

Seasonal and individual variation in singing behavior correlates with alpha 2-noradrenergic receptor density in brain regions implicated in song, sexual, and social behavior

S A Heimovics a,1, C A Cornil b,c, J M S Ellis a, GF Ball c, LV Riters a,*
PMCID: PMC3085591  NIHMSID: NIHMS281369  PMID: 21397668

Abstract

In seasonally breeding male songbirds, both the function of song and the stimuli that elicit singing behavior change seasonally. The catecholamine norepinephrine (NE) modulates attention and arousal across behavioral states, yet the role of NE in seasonally-appropriate vocal communication has not been well-studied. The present study explored the possibility that seasonal changes in alpha 2-noradrenergic receptors (α2-R) within song control regions and brain regions implicated in sexual arousal and social behavior contribute to seasonal changes in song behavior in male European starlings (Sturnus vulgaris). We quantified singing behavior in aviary housed males under spring breeding season conditions and fall conditions. α2-R were identified with the selective ligand [3H]RX821002 using autoradiographic methods. The densities of α2-R in song control regions (HVC and the robust nucleus of the arcopallium [RA]) and the lateral septum (LS) were lower in Spring Condition males. α2-R densities in the caudal portion of the medial preoptic nucleus (POM) related negatively to singing behavior. Testosterone concentrations were highest in Spring Condition males and correlated with α2-R in LS and POM. Results link persistent seasonal alterations in the structure or function of male song to seasonal changes in NE α2-Rs in HVC, RA, and LS. Individual differences in α2-R in the POM may in part explain individual differences in song production irrespective of the context in which a male is singing, perhaps through NE modification of male sexual arousal.

Keywords: communication, seasonality, norepinephrine, song control system, social behavior, motivation

1. Introduction

To communicate effectively animals must attend to specific stimuli within the environment and respond with social signals that are appropriate within a particular context and reflective of the animal’s internal or endocrine state. In seasonally breeding songbirds that sing throughout the year, the function of song changes seasonally (Marler and Slabbekoorn, 2004; Catchpole and Slater, 2008). During the breeding season (e.g., spring) circulating concentrations of sex steroids are elevated (Wingfield and Farner, 1993; Wingfield, 2006). At this time males are aroused by, direct attention towards, and sing at high rates to attract females or to repel competing males (Catchpole and Slater, 2008). In contrast, outside of the breeding season (e.g., in fall and winter), sex steroid concentrations are basal. At this time, in some species males continue to sing at high rates, but they do not clearly direct song towards or adjust song in response to other individuals. Song in this context likely plays a role in song learning and may function to keep flocks together (e.g., (Feare, 1984; Eens, 1997). Studies of songbirds have provided crucial insights into specific brain regions involved in the learning, production, and auditory processing of vocal behavior (for recent reviews see (Ball et al., 2008; Brainard, 2008; Brenowitz, 2008; Gentner, 2008; Nordeen and Nordeen, 2008; Theunissen et al., 2008; Wild, 2008); however, little is known about how the brain regulates vocal communication so that it occurs within an appropriate seasonal context.

Across vertebrate species, the catecholamine norepinephrine (NE) plays a role in modulating optimal behavioral responses, sensory responsiveness, attention, and arousal across behavioral states (Berridge and Waterhouse, 2003; Aston-Jones and Cohen, 2005; Castelino and Schmidt, 2009). In songbirds, nuclei of the song control system contain NE receptors and synthesizing enzymes (Mello et al., 1998; Riters and Ball, 2002a; Riters et al., 2002), and NE modifies neuronal firing and/or immediate early gene expression in several song control nuclei (Cardin and Schmidt, 2004; Castelino and Ball, 2005; Solis and Perkel, 2006; Sizemore and Perkel, 2008). Furthermore, the boundaries of several nuclei (including HVC [used as a proper name] and the robust nucleus of the arcopallium [RA]) are well-defined by the presence of α2-Rs (Bernard and Ball, 1995; Riters and Ball, 2002a; Riters et al., 2002).

Past work in songbirds implicates NE in male courtship singing in response to females. For example, in male zebra finches NE levels within some song control nuclei correlated positively with courtship singing (Barclay et al., 1992), and NE depletion using the neurotoxin DSP-4 decreased the numbers of song bouts performed and increased the latency for males to initiate song in response to a female (Barclay et al., 1996). In starlings, α2-noradrenergic receptor (α2-R) densities in song control regions (HVC and RA) were lower in males trapped in spring (when testosterone was high and males sing to attract females or repel males) compared to males trapped in fall (when testosterone was low and song functions to maintain flocks) (Riters et al., 2002). Together these data highlight a role for NE in song production and show that shifts in NE receptor densities in song control regions accompany seasonal shifts in the function of song.

In male songbirds, data suggest that several NE rich regions outside of the song control system (Mello et al., 1998; Riters and Ball, 2002b), including the medial preoptic nucleus (POM) and lateral septum (LS), are differentially involved in season-specific singing behavior (Goodson et al., 1999; Heimovics and Riters, 2005; 2006; 2007; Heimovics et al., 2009). Together these data suggest that in addition to its direct action on the song system, NE may also influence singing behavior through its action on brain areas implicated in sexual motivation and social behavior.

Short term changes in behavioral state (such as sleep versus wakefulness) are regulated by rapid alterations in NE release (Berridge and Waterhouse, 2003; Castelino and Schmidt, 2009). Perhaps longer-term changes, such as persistent seasonal alterations in male sexual arousal and the production of seasonally-appropriate song may in part be explained by seasonal changes in NE receptors. The present study used receptor autoradiography to provide insight into this possibility by examining song production and α2-R densities in male European starlings experiencing Spring-like and Fall-like Conditions.

2. Experimental procedures

2.1 Starling capture and housing

Thirty experimental male and ten stimulus female European starlings were captured using fly-in traps between December 2005 and February 2006 on a farm near Madison, Wisconsin. Birds were then housed in single sex groups indoors in stainless steel cages (91 × 47 × 47 cm) in the University of Wisconsin – Madison Department of Zoology animal facilities. Birds were placed on a photoperiod of 18 hours light (L): 6 hours dark (D) for six weeks, followed by 6 weeks on a photoperiod of 6L:18D. Male starlings exposed to these shifts in photoperiod become “photosensitive”, a condition observed prior to the onset of the breeding season in which males respond to increases in day length with gonadal recrudescence and increased T production (Dawson et al., 2001). All protocols were approved by the University of Wisconsin Institutional Animal Care and Use Committee and adhered to methods approved by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

2.2 Hormone treatment

In a previous field study, α2-R density in the song system differed in males that were captured at a time of year at which they were photosensitive with low T (outside the breeding season) as compared to photostimulated with high T (within the breeding season (Riters et al., 2002)). In the present study, T and photoperiod manipulations were used to simulate these conditions. To simulate the breeding season, 20 photosensitive males (now referred to as the “Spring Condition” group) received 2 subcutaneous Silastic T-containing implants (14 mm length, i.d. 1.47 mm; o.d. 1.96 mm [Dow Corning, Midland, MI, USA], packed for 10 mm with crystalline T propionate and sealed with Silastic glue [Sigma Aldrich, St. Louis, MO, USA]) and were shifted to 11L:13D (a photoperiod which induces gonadal growth characteristic of the early breeding season (Dawson et al., 2001)). To simulate the non-breeding season, 10 photosensitive males (now referred to as the “Fall Condition” group) received 2 empty Silastic implants and remained on 6L:18L. During implant surgery, birds were anesthetized using isoflurane gas anesthesia and a small incision was made in the skin over the breast muscle. Implants were placed under the skin and the wound was sutured. After recovering on a heated pad, birds were placed back into single-sex cages.

2.3 Behavioral observations

One to two days following implant surgery two flocks of 10 Spring Condition males and one flock of 10 Fall Condition males were placed into separate indoor behavioral observation aviaries. Each aviary contained five nest boxes and branches for perching. Food and water were available ad libitum. Males were individually color banded for identification. In Spring Condition some males will occupy nest boxes whereas others will not. Although not the focus of this study, we were initially interested in examining α2-R densities in both males with and without nest boxes; therefore, we examined two flocks of Spring Condition males to ensure our sample included adequate numbers of males with and without nest boxes. Behavioral observations began after a 4-week habituation period and took place between 09:00 and 12:00h.

Flocks were observed on five consecutive days for 30 min on each day. During this time, a point sampling technique was used to determine the proportion of time any member of the flock spent singing (i.e. during each 30-min observation period it was noted at 60-s intervals whether and from where any member of the flock was singing). For purposes that are not the focus of the present paper, both Spring and Fall Condition males were also presented with a female for 30 min each day (an estradiol implanted female for Spring Condition males and a blank (empty) implanted female for Fall Condition males). The order of 30-min observations (with versus without a female present) was counterbalanced across days, and a novel female conspecific was released into the aviary each day.

2.4 Tissue processing

One day after completion of behavioral observations, males were rapidly decapitated and brains collected. For every male, approximately 2mL of trunk blood were collected and centrifuged at 3000G for 30min at 4°C. Blood plasma (supernatant) was then collected and stored at −20°C until assayed for T. Brains were removed, frozen immediately on powdered dry ice, and stored at −80 °C until sectioning. Brains were sectioned using a cryostat. Sections sixteen microns thick were thaw mounted onto gel-coated microscope slides. Six series of slides were collected so that, on each slide, consecutive sections were 80 microns apart. One series was used for α2-R autoradiography, and one series was Nissl stained to aid in the identification of brain regions in which α2-R were measured in the study reported here. Other series were used for a previously published dopamine D1-like and D2-like receptor autoradiography (Heimovics et al., 2009). The slides were dried and stored at −20 °C until use.

2.5 Autoradiography

α2-R were identified by using the highly selective α2-R antagonist, [3H]RX821002, which has been described as a radioligand of choice to detect the total population of α2-R in several tissues and species, including birds (O’Rourke et al., 1994; Halme et al., 1995; Ruuskanen et al., 2005; Diez-Alarcia et al., 2006; Cornil and Ball, 2008). After drying at room temperature, slides were pre-incubated in buffer (50 nM Tris-HCl, pH 7.5 at 25°C with 1mM MgCl2) for 30 min at room temperature. Slides were then incubated for one hour at room temperature in 5nM [3H]RX821002 (specific activity: 49.0 Ci/mmol; Amersham Biosciences, Piscataway, NJ, USA) buffer. In parallel, a few slides were incubated in 5nM [3H]RX821002 to which was added phentolamine-HCl 10 μM (obtained from Sigma-Aldrich, Inc.) to determine the non-specific binding of this ligand. One hour later, the slides were washed twice for five min in ice-cold buffer followed by a quick dip in ice-cold distilled water. Sections were then fan dried, placed in X-ray cassettes and exposed to tritium-sensitive BioMax® MR films (Kodak) along with standards (ART-123; American Radiolabeled Chemicals Inc., St Louis, MO) containing concentrations of tritium ranging from 0.00 to 489.1 μCi/g. Sections were run in two batches and tissue from birds in different groups was arbitrarily distributed across batches and film cassettes. The films were developed after 12 weeks.

2.6 Quantification

Each of the developed films was scanned (eight-bit, 600 dpi) using an Epson Perfection 1240 U bed scanner connected to a PC computer. Digitized autoradiograms were analyzed using MetaVue software (Fryer Company, Inc., Huntley, IL, USA) following standard procedures (e.g. (Wang et al., 1997; Riters et al., 2002; Chen and Lawrence, 2003). Specifically, the tritium standards were used to calibrate the intensity of the scanned images in terms of radiochemical concentrations in fmol/mg using the following equation: (X microCi/1000 mg)*(106 fmol/Y mg), where X equals the numerical values associated with the standard and Y equals the specific activity of the ligand (49 Ci/mmol). The gray level value of each standard was then measured in MetaVue and the known concentration of radioactivity (in fmol/mg) was assigned to each corresponding gray level. Values for standards spanning the range of optical density of labeled tissue were selected, and the relationship between the gray values and measures of radioactivity was fit to a 3rd degree polynomial equation, which was used to interpolate gray values between points on the standards. Using these calibrated gray levels, the average intensity of specific binding for α2-R was determined for two song control regions, HVC and RA, and two regions implicated in social behavior, the POM and LS. In order to more effectively demonstrate any regional specificity of group differences in ligand binding, we additionally measured α2-R density in two control regions for which we had no a priori reasons to predict seasonal differences. The first was area X, a song control region in which α2-Rs were not found to change seasonally in our past work (Riters et al., 2002) and the bed nucleus of the stria terminalis, a region involved in social behavior (e.g.,(Goodson, 2005)) for which we found no literature that would lead us to hypothesize that α2-Rs would change seasonally. Locations of each nucleus were identified based on (Heimovics and Riters, 2007); Figure. 1) and verified using landmarks visible on both autoradiograms and/or adjacent Nissl-stained sections.

Figure 1.

Figure 1

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Illustrations of coronal sections from one hemisphere of the starling brain. Sections A–E progress from rostral to caudal. Circles indicate approximate areas in which α2-R densities were quantified. Abbreviations: A, arcopallium; BST, bed nucleus of the stria terminalis; Cb, cerebellum; CO, optic chiasm; CoA, anterior commissure; GCt, mesencephalic central gray; HA, apical part of the hyperpallium; HD, densocellular part of the hyperpallium; MSt, medial striatum; LS, lateral septum; mMAN, medial magnocellular nucleus of the anterior nidopallium; MS medial septum; NIII, third cranial nerve; N, nidopallium; NC, caudal nidopallium; POM, medial preoptic nucleus; RA, robust nucleus of the arcopallium; Rt, nucleus rotundus; TnA, nucleus taeniae of the amygdala; V, ventricle; VTA, ventral tegmental area.

Typically, specific binding is determined by subtracting non-specific binding values from total binding values. However in this experiment (as in past studies examining on α2-Rs in songbirds [e.g., (Riters et al., 2002)]), no non-specific labeling was observed in any region in sections treated with phentolamine, indicating that the labeling observed was specific to α2-R. Within each region, total binding was quantified by measuring the average intensity of calibrated gray levels within a circular region located within the boundaries of each nucleus (visible in adjacent Nissl stained tissue) on 3 serial sections bilaterally. In cases of tissue damage or uneven exposure of autoradiograms, average intensity was measured on a fourth section. If tissue damage and/or uneven exposure was extensive, that individual was dropped from quantification for that brain area. For all individuals, specific binding values were averaged separately for each region and the mean used for statistical analysis.

Additional quantification was performed for both HVC and POM based on past work showing HVC α2-R binding densities to differ along the rostro-caudal extent (Riters et al., 2002) and data indicating distinct functions for rostral and caudal portions of POM in male sexual behavior (Balthazart et al., 1998; Riters et al., 2004; Taziaux et al., 2006; Balthazart and Ball, 2007). Specifically, α2-R binding densities were quantified within 3 subdivisions of HVC and 4 subdivisions of POM. For HVC, bilateral measurements were made in the most rostral, intermediate, and caudal sections of HVC. Measures were taken from 3 sequential sections within each subdivision of HVC. For POM, bilateral measurements were made in sections in the most rostral portion of POM (just ventromedial to the tractus septopalliomesencephalicus [TSM]), in rostral-intermediate POM (just posterior to the level of the full extension of TSM), in caudal-intermediate POM (just prior to the appearance of the anterior commissure [AC]) and in the caudal POM (ventral to the AC). Measures were taken from 3 serial sections within each subdivision of POM. Analyses were performed on the mean of each subdivision of HVC and POM and the mean of the measurements taken for the entire nucleus.

2.7 Testosterone Assay

Plasma testosterone was measured using a commercially available enzyme immunoassay kit (Cayman Chemical 582701) used to assay T in multiple songbird species (e.g., (Pryke et al., 2007; Gil et al., 2008; Sughrue et al., 2008; McGuire and Bentley, 2010; Kelm et al., in press)). Samples were run in duplicate with respect to a standard curve (limit of detection: 80% B/B0: 6 pg/ml; sensitivity: 50% B/B0: 32 pg/ml). The within assay coefficient of variation was 6.87%. A set of identical internal standards was run in each assay. The antibody used in this assay is highly specific for testosterone but has some cross-reactivity with other androgens (5-α DHT: 27.4%; A4:3.7%).

2.8 Statistical treatment of the data

Paired comparisons, t-tests and where appropriate Mann-Whitney U tests, indicated that α2-R densities did not differ significantly in any brain region in males with and without nest boxes, thus for all analyses these animals were combined to form the Spring Condition. The mean proportion of min at which each male was singing (described in methods above) on each of the 5 test days was calculated. Means were then arcsine transformed as appropriate for proportion data (Lehner, 1996). Multiple regression analyses were then performed to examine the extent to which male condition (Fall or Spring) and song production contributed statistically to variation in α2-R densities in the focal regions HVC, RA, LS and POM and additionally the two control regions area X and the bed nucleus of the stria terminalis.

A separate set of analyses were run to examine the possibly functionally distinct subdivisions of HVC and POM (as suggested by (Balthazart et al., 1998; Riters et al., 2002; Riters et al., 2004; Taziaux et al., 2006)). Repeated measures ANOVAs were used to compare α2-R densities across subdivisions of HVC and POM, with significant differences followed by Fisher LSD post hoc tests. Multiple regression analyses were also performed to examine the contribution of male condition and song production to α2-R densities in subdivisions of HVC and POM.

Testosterone data for Fall Condition males were not normally distributed therefore comparisons between Spring and Fall Condition males were made using non-parametric Mann-Whitney U tests. Spearman correlations were also used to examine relationships between testosterone and α2-R densities for birds in Spring and Fall Conditions combined. For the testosterone analyses, data were dropped for 4 individuals for which samples were contaminated with red blood cells (1 Fall Condition, 3 Spring Condition).

For all analyses differences in sample sizes reflect missing data due to tissue damage. For multiple regression analyses the results of both forward and backward stepwise analyses were identical for all brain regions except in the analysis of LS. For LS, forward and backward analyses yielded the same significant effects and we report the results of forward regression based on the higher R2 and lower standard error.

3. Results

3.1 Contribution of condition and song production to α2-R densities

Results of a multiple regression analysis revealed a significant contribution of male condition (Spring versus Fall) but not song production to mean HVC α2-R density (regression results: n = 26, adj R2 = 0.18, p = 0.019, significant contribution of male condition: beta = 0.46, beta se = 0.18) with denser HVC α2-R observed in Fall compared to Spring Condition males (Figure 2 and 3). Similar results were obtained for α2-R density in RA (regression results: n = 27, adj R2 = 0.22, p = 0.008, significant contribution of male condition: beta = 0.50, beta se = 0.18; Figure 2 and 3) and in LS (regression results: n = 25, adj R2 = 0.23, p = 0.009, significant contribution of male condition: beta = 0.51, beta se = 0.18; Figure 2 and 3). No significant contributing variables were identified in α2-R densities for mean POM, area X, or the bed nucleus of the stria terminalis.

Figure 2.

Figure 2

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Representative photomicrographs showing densities of α2-R in HVC (top), RA (middle), and LS (bottom) in a Fall Condition (left) and Spring Condition (left) male. Photomicrographs were scanned and imported into Adobe Photoshop Elements 6.0. The contrast for each image was enhanced identically for all individuals being compared.

Figure 3.

Figure 3

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Bar graphs illustrating the mean (+sem) α2-R densities in HVC (the mean for all sections quantified), RA, and LS. Open bars represent Fall Condition males. Filled bars represent Spring Condition males. Dots overlying each bar represent the mean α2-R density measure for each individual bird. Sample sizes are indicated in each bar. * = p < 0.05.

3.2 Mean α2-R densities in subdivisions of HVC and POM

A repeated measures ANOVA indicated that α2-R density increases in a rostral to caudal direction in HVC (mean density [sd]: rostral HVC, 86.07 [14.17]; intermediate HVC, 101.62 [23.40]; caudal HVC, 133.04 [33.36]; F2,42 = 62.11, p < 0.001, with Fisher LSD post hoc tests revealing significant differences between each subdivision). When Breeding Condition was included as a factor, significant differences in α2-R densities were observed across subdivisions (F2,40 = 55.60, p < 0.001). Both Breeding Condition (F 1,20 = 4.03, p = 0.058) and the subdivision by breeding condition interaction (F 2,40 = 2.58, p = 0.088) approached but did not reach statistical significance.

A repeated measures ANOVA indicated that α2-R density is highest within intermediate portions of POM (mean density [sd]: rostral POM, 242.57 [48.63]; rostral-intermediate POM, 305.80 [62.07]; caudal-intermediate POM, 293.46 [66.56]; caudal POM, 242.42 [51.52]; F 3,75 = 50.68, p < 0.001, with Fisher LSD post hoc tests revealing significant differences between both of the intermediate subdivisions and rostral POM as well as caudal POM). When Breeding Condition was included as a factor, significant differences in α2-R densities were observed across subdivisions (F 3,72 = 42.25, p < 0.001); however, neither Breeding Condition nor the interaction were significant (p > 0.62 in each case).

3.3 Contribution of condition and song production to α2-R densities in HVC and POM subdivisions

Results of multiple regression analyses revealed a significant contribution of male condition (Spring versus Fall) but not song production to α2-R densities in both intermediate and caudal portions of HVC (intermediate HVC regression results: n = 26, adj R2 = 0.20, p = 0.012, significant contribution of male condition: beta = 0.48, beta se = 0.18; caudal HVC regression results: n = 24, adj R2 = 0.15, p = 0.033, significant contribution of male condition: beta = 0.44, beta se = 0.19) with denser HVC α2-R observed in Fall compared to Spring Condition males. No significant contributors were identified for α2-R density in rostral HVC.

For both intermediate and caudal POM, song production but not male condition statistically contributed to variation in α2-R density (intermediate POM regression results: n = 30, adj R2 = 0.11, p = 0.044, significant contribution of song production: beta = −0.37, beta se = 0.18; caudal POM regression results: n = 30, adj R2 = 0.17, p = 0.013, significant contribution of song production: beta = −0.45, beta se = 0.17; Figure 4). No significant contributors were identified for α2-R density in rostral or rostral intermediate POM.

Figure 4.

Figure 4

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Scatterplot illustrating significant contribution of singing behavior to α2-R densities in caudal POM. Untransformed data are shown to illustrate actual values. Analyses were performed on arcsine transformed difference scores (see text for details). Each circle represents an individual bird. Open circles = Fall Condition males; Filled circles = Spring Condition males.

3.4 Testosterone (T)

A Mann-Whitney U test comparing Fall Condition (blank implanted; n = 9) and Spring Condition (T implanted; n = 17) males verified the effects of T implants. T was significantly higher in Spring compared to Fall Condition males (MWU = 5.0, z = 3.85, p = 0.0001; Spring mean = 1539.96 pg/mL, sd = 711.68; Fall mean = 153.86 pg/mL, sd = 436.15 [2 of 9 males had detectable T]). Correlation analyses revealed a significant negative correlation between T and α2-R densities in LS (r = −0.56, p = 0.009; Figure 5). A positive relationship was found between T and α2-R densities for the mean of all subdivisions of POM (r = .53, p = 0.006; Figure 5). The results were similar for each of the subdivisions of POM (rostral POM, r = 0.51, p = 0.014; rostral-intermediate POM, r = 0.47, p = 0.028; caudal-intermediate POM, r = 0.43, p = 0.033; caudal POM r = 0.52, p = 0.008). These correlations were influenced by the Fall Condition males who had low to undetectable T values. When only birds in Spring Condition were examined no significant correlations were found between T and α2-R densities. No other significant correlations were identified for any of the brain regions examined when all males were considered together or divided according to condition.

Figure 5.

Figure 5

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Scatterplots illustrating significant correlations between testosterone concentrations and α2-R densities in LS and POM. Each circle represents an individual bird. Open circles = Fall Condition males; Filled circles = Spring Condition males.

4. Discussion

The results of the present study link alterations in α2-R densities in regions within and outside of the song control system to seasonally appropriate or individual differences in male song production. Determining directional and causal relationships between seasonal changes in α2-R densities and song production awaits future direct manipulations; however the present results provide a strong step toward understanding seasonal changes in α2-Rs and ways in which these changes may contribute to changes in singing behavior.

4.1 Functional role of condition-dependent variation in α2-R densities

α2-R densities were lower in Spring Condition compared to Fall Condition males in song control regions HVC and RA and a region implicated in sexual and social behavior, LS. The findings for HVC and RA are consistent with past studies of male starlings experiencing natural photoperiods and T fluctuations in the wild, with elevated T and a longer photoperiod associated with low density α2-R in both HVC and RA (Riters et al., 2002). HVC and RA are important for the arrangement of temporal units of song (Margoliash, 1997). In male starlings structural aspects of song, including bout length and repertoire size have been found to increase in spring (Riters et al., 2000; Van Hout et al., 2009), and in other songbird species measures of song stereotypy and numbers of learned syllables change seasonally in association with season and changes in the function of song (e.g., (Nottebohm, 1981; Nottebohm et al., 1981; Smith et al., 1997a)). Based on previous studies, NE stimulation of α2-R in male zebra finches reduced spontaneous activity in RA (Solis and Perkel, 2006) and reduced input to RA from LMAN (Sizemore and Perkel, 2008), which is a region implicated in context-specific adult song variability (Kao and Brainard, 2006; Kao et al., 2008). α2-Rs are found both pre- and post-synaptically, with the pre-synaptic receptors functioning as auto-receptors, inhibiting NE release (Starke, 2001). Thus, down-regulation of α2-R within HVC and RA of Spring Condition males may function to enhance overall NE activity to directly (or perhaps indirectly by influencing another neurochemical systems (Sizemore and Perkel, 2008)) stimulate structurally appropriate features of song used to attract a female or repel a male.

Although not a “song control” nucleus, data suggest LS context-dependently modifies male song production. Lesions to LS can stimulate or inhibit courtship song depending upon whether the species examined is territorial or gregarious (Goodson et al., 1999), and activity in LS as indicated by immediate early gene markers differs in male starlings singing in Spring and Fall Conditions (Heimovics and Riters, 2006; 2007). The present data suggest alterations in α2-Rs in LS may play a role in altering LS regulation of song seasonally. Furthermore, α2-Rs in LS inhibit male sexual arousal in rats (Gulia et al., 2002), thus downregulation of α2-R within LS of Spring Condition males may function to facilitate seasonally appropriate sexual responses to females, including sexually-motivated song in spring.

4.2 α2-R densities in POM relate to song production but not seasonal condition

In contrast to the patterns observed for HVC, RA, and LS, our data indicate that song production rather than male condition best predicted statistical variation in α2-R densities in both intermediate and caudal portions of POM. Several studies implicate the POM in sexually-motivated male song production in Spring Condition starlings (Riters and Ball, 1999; Heimovics and Riters, 2005; Alger and Riters, 2006; Alger et al., 2009). A growing number of studies also indicate that the role of the POM extends to song in Fall Condition males (Riters et al., 2005; Alger and Riters, 2006; Heimovics et al., 2009) and song used to defend nesting territories from other males in Spring Condition birds (Heimovics et al., 2009). Past work in male zebra finches shows that NE depletion using the neurotoxin DSP-4 substantially reduced NE in the medial preoptic area (as well as other areas) and resulted in a delay in the initiation of singing behavior (Barclay et al., 1996). These findings were interpreted as reflecting a role for NE in arousal or attention needed to initiate song production. The POM is critically involved in male sexual arousal across vertebrates (Crews, 2005; Balthazart and Ball, 2007; Hull and Dominguez, 2007; Ball and Balthazart, 2010), and studies in quail suggest that NE in the POM inhibits male sexual behavior (Bailhache et al., 1993). It is thus possible that increasing α2-R densities (which may result in a reduction in NE activity, through pre-synaptic α2-auto-receptor inhibition (Starke, 2001)) in caudal POM may underlie seasonally appropriate increases in sexual behavior, including sexually-motivated song. Indeed although seasonal condition did not contribute significantly to statistical variation in α2-R densities, Spring Condition males generally had higher densities of α2-R in caudal POM than Fall Condition males (Figure 4). Thus by modifying an individual’s state of sexual arousal, individual differences in α2-R densities in POM may underlie individual differences in song production.

4.3 The role of testosterone in condition-dependent differences in α2-R densities

As expected, T was significantly elevated in Spring (T implanted) compared to Fall (blank implanted) Condition males. Thus the seasonal changes in α2-R in HVC, RA, and LS may be modified directly by seasonal fluctuations in T. In support of this idea, in the present study T concentrations correlated with α2-R densities in LS and POM; however these correlations were not observed when males in Fall or Spring Condition (the condition in which T varied most) were considered alone. Correlations between T and α2-R densities were not found for HVC or RA; however, it is possible that T induced alterations in α2-R are not linear for these regions, but that once T crosses a certain threshold α2-R densities drop. Links between T and noradrenergic systems have been documented in several past studies in birds (Harding et al., 1983; Barclay and Harding, 1988; Balthazart and Ball, 1989; Ball and Balthazart, 1990; Barclay and Harding, 1990). HVC, RA, LS, and caudal POM all contain receptors for androgens or androgen metabolites (Gahr, 2001). The extent to which T plays a direct role in altering α2-R densities in these regions must be determined in future research.

T also plays an important role in mediating seasonal changes in neural plasticity of the song control system (Ball et al., 2004; Brenowitz, 2004; Meitzen and Thompson, 2008). T treatment increases the volumes of HVC and RA (Tramontin et al., 2003), whereas castration rapidly reduces the volumes of these regions (Thompson et al., 2007). Given the seasonal expansion and contraction of HVC and RA it may be that as the volume of a nucleus increases this causes a stable population of receptors to spread out resulting in decreased density; however, past work in male starlings does not support this possibility. Specifically, increases in the volumes of HVC and RA were not accompanied by proportionally equivalent decreases in α2-R density (Riters et al., 2002). Furthermore, changes in volume and density do not occur synchronously, rather increases in volume begin earlier in spring than decreases in α2-R density (Riters et al., 2002). Although not extensively studied, the volume of LS has not been found to change seasonally in non-food storing birds (Shiflett et al., 2002). Thus differences found between Fall and Spring Condition males in α2-R densities in HVC, RA, as well as LS likely reflect seasonal changes in receptor numbers or binding affinity, but this must be determined in future work.

4.4 The role of photoperiod in condition-dependent alterations in α2-R densities

Similar to birds living in the wild, the birds in Spring and Fall Condition in the present study differed not only in testosterone concentrations but also with respect to the photoperiods to which they were exposed (11L:13D for Spring Condition males and 6L:18D for Fall Condition males). Past studies in songbirds indicate that small seasonal changes in the volumes of HVC and RA can occur independently from T concentrations (Smith et al., 1997b; Dloniak and Deviche, 2001; Tramontin et al., 2001; Riters et al., 2002; Caro et al., 2005). Thus, in the present study, as well as in the wild, differences in the photoperiods to which Spring and Fall Condition males were exposed may have contributed to differences in receptor density.

4.5 α2-R densities vary regionally within HVC and POM

Similar to past results, α2-Rs were denser in rostral than caudal HVC (Riters et al., 2002). Furthermore, differences in α2-density in Spring and Fall Condition males were restricted to intermediate and caudal portions of HVC. This suggests that HVC may possess functionally distinct rostral and caudal subdivisions and that seasonal alterations in α2-R specifically in intermediate and caudal HVC are particularly important for seasonal adjustments of song structure.

α2-R densities also differed along the rostrocaudal axis in POM, with the densest receptor concentrations restricted to intermediate rather than rostral or caudal POM. These findings are consistent with previous studies highlighting distinct subdivisions within POM. For example, both results of lesion studies and studies using immediate early genes suggest that the rostral POM is more critical for the regulation of sexual interest and that the caudal portion is critical for copulation (Balthazart et al., 1998; Riters and Ball, 1999; Riters et al., 2004; Taziaux et al., 2006). The present data showing that α2-R in intermediate and caudal POM but not rostral or rostral-intermediate POM contributed to statistical variance in male song production offer further support to prior work identifying distinct functional topography within the POM (reviewed in (Balthazart and Ball, 2007)).

4.6 α2-R densities did not relate to condition or song in all regions

α2-R densities in area X and the bed nucleus of the stria terminalis did not correlate with song behavior, testosterone or seasonal condition. These regions were not expected to display seasonal changes in α2-R (i.e., past studies do not suggest seasonal changes in α2-Rs in these areas). They were included as a control to assess the extent to which group differences in ligand binding were regionally specific. The failure to find changes as a function of condition for these regions indicates that differences observed in HVC, RA, and LS as well as correlations between song and POM are confined to select regions for which we had a priori reasons to predict a role for NE in song or social behavior (as reviewed in the introduction).

4.7 Conclusions

The links between α2-R and brain regions involved in song control and social behavior are consistent with the idea that persistent seasonal alterations in the structure or function of male song may in part be explained by seasonal changes in NE α2-Rs in HVC, RA, and LS. The results of this study also identify for the first time a context-independent linear relationship between song and α2-R in the POM, suggesting that individual differences in a male’s propensity to sing within any context may be regulated by α2-R activity within this region, perhaps through NE modification of male sexual arousal. This finding more broadly contributes to the understanding of the mechanisms underlying consistent individual variation in behavior (or “personality”), a poorly understood topic that is receiving increasing attention (e.g., (Ball and Balthazart, 2008; Williams, 2008; Duckworth, 2010)). Altogether these findings are consistent with the hypothesis that seasonal alterations and individual differences in α2-R densities in regions within and outside of the song control system may modulate NE activity to support seasonally appropriate or individual differences in singing behavior; however, the precise causal relationships among these variables and specific contributions of NE activity within each region to behavior await future research.

Acknowledgments

This work is supported by grant R01 MH080225 to LVR, grant R01 NS 35467 to GFB, and an NSF pre-doctoral fellowship to SAH. CAC is a F.R.S.-FNRS Research Associate. Additionally, we thank Sharlene Shu and Sharon Stevenson for technical assistance.

Footnotes

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Contributor Information

S. A. Heimovics, Email: saheimovics@psych.ubc.ca.

C. A. Cornil, Email: charlotte.cornil@ulg.ac.be.

J. M. S. Ellis, Email: jmellis2@wisc.edu.

G.F. Ball, Email: gball@jhu.edu.

L.V. Riters, Email: LVRiters@wisc.edu.

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