Hormones and Behavior 69 (2015) 31–38

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Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh

Regular article

Non-invasive administration of 17β-estradiol rapidly increases aggressive behavior in non-breeding, but not breeding, male song sparrows Sarah A. Heimovics a,b,⁎, Jennifer K. Ferris c,e, Kiran K. Soma c,d,e a

Department of Biology, University of St. Thomas, St. Paul, MN, USA Neuroscience Program, University of St. Thomas, St. Paul, MN, USA c Department of Psychology, University of British Columbia, Vancouver, BC, Canada d Department of Zoology, University of British Columbia, Vancouver, BC, Canada e Graduate Program in Neuroscience, University of British Columbia, Vancouver, BC, Canada b

a r t i c l e

i n f o

Article history: Received 4 June 2014 Revised 25 November 2014 Accepted 27 November 2014 Available online 4 December 2014 Keywords: Non-genomic Nongenomic Rapid Estrogen Estrogens Brain Aggression Season Bird Songbird

a b s t r a c t 17β-Estradiol (E2) acts in the brain via genomic and non-genomic mechanisms to influence physiology and behavior. There is seasonal plasticity in the mechanisms by which E2 activates aggression, and non-genomic mechanisms appear to predominate during the non-breeding season. Male song sparrows (Melospiza melodia) display E2-dependent territorial aggression throughout the year. Field studies show that song sparrow aggression during a territorial intrusion is similar in the non-breeding and breeding seasons, but aggression after an intrusion ends differs seasonally. Non-breeding males stop behaving aggressively within minutes whereas breeding males remain aggressive for hours. We hypothesize that this seasonal plasticity in the persistence of aggression relates to seasonal plasticity in E2 signaling. We used a non-invasive route of E2 administration to compare the nongenomic (within 20 min) effects of E2 on aggressive behavior in captive non-breeding and breeding season males. E2 rapidly increased barrier contacts (attacks) during an intrusion by 173% in non-breeding season males only. Given that these effects were observed within 20 min of E2 administration, they likely occurred via a non-genomic mechanism of action. The present data, taken together with past work, suggest that environmental cues associated with the non-breeding season influence the molecular mechanisms through which E2 influences behavior. In song sparrows, transient expression of aggressive behavior during the non-breeding season is highly adaptive: it minimizes energy expenditure and maximizes the amount of time available for foraging. In all, these data suggest the intriguing possibility that aggression in the non-breeding season may be activated by a non-genomic E2 mechanism due to the fitness benefits associated with rapid and transient expression of aggression. © 2014 Elsevier Inc. All rights reserved.

Introduction 17β-estradiol (E2) acts in the brain via both genomic and nongenomic signaling mechanisms to influence physiology and behavior (Vasudevan and Pfaff, 2008). In the genomic model of steroid action, E2 binds to cytosolic estrogen receptors (ERs), and the hormonereceptor complex translocates to the cell nucleus, and binds to estrogen response elements in the DNA to alter gene expression (Jensen et al., 1968; McCarthy, 2009; Vasudevan and Pfaff, 2008). These effects generally take several hours or days to develop (Zangenehpour and Chaudhuri, 2002), and lead to persistent changes in physiology and behavior (McCarthy, 2009; McEwen, 2001). However, E2 also acts on a

⁎ Corresponding author at: University of St Thomas, USA. E-mail address: [email protected] (S.A. Heimovics).

http://dx.doi.org/10.1016/j.yhbeh.2014.11.012 0018-506X/© 2014 Elsevier Inc. All rights reserved.

timescale that is too short to be attributed to changes in gene transcription (Cornil and Charlier, 2010). In this non-genomic model of E2 action, E2 binds to plasma membrane-associated ERs, which activate signal transduction cascades including mobilization of cytosolic calcium and phosphorylation of cAMP response element binding (CREB) and mitogen-activated protein kinase (MAPK) (Ivanova et al., 2002; Kelly et al., 1999; Singer et al., 1999). These rapid, non-genomic effects typically occur within minutes and lead to more transient changes in physiology and behavior (Laredo et al., 2014). Recent data suggest that the signaling mechanisms by which E2 regulates aggressive behavior are modulated by photoperiod. Specifically, in Peromyscus mice, acute E2 administration rapidly alters aggressive behavior in male subjects housed on short (non-breeding season-like) photoperiods but not those housed on long (breeding season-like) photoperiods (Trainor et al., 2007a, 2008). Further, microarray and realtime PCR analyses indicate that estrogen response element-dependent

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S.A. Heimovics et al. / Hormones and Behavior 69 (2015) 31–38

gene expression is higher in animals housed on long photoperiods as compared to those housed on short photoperiods (Trainor et al., 2007a). Taken together, these data indicate that the non-genomic effects of E2 on aggressive behavior may be more prominent during the non-breeding season, whereas the genomic effects of E2 on aggression may be more prominent during the breeding season (Trainor et al., 2007a). Like Peromyscus, male song sparrows (Melospiza melodia) display E2-dependent territorial aggression throughout the year (except for a brief period during molt) (Wingfield and Soma, 2002). Territorial aggression in song sparrows is measured in the field via simulated territorial intrusion (STI) whereby a live caged decoy and conspecific song playback are used to elicit aggressive behavior in residents (Soma et al., 2000). Territorial aggression during a STI is both qualitatively and quantitatively similar in the breeding and non-breeding seasons (Wingfield and Hahn, 1994). However, territorial aggression after a STI is terminated changes seasonally. In the breeding season, residents continue patrolling their territories and exhibit spontaneous song for hours (even days) after a STI, whereas in the non-breeding season, residents stop behaving aggressively within minutes (Wingfield, 1994). It appears that once the behavior is elicited, breeding territorial aggression is persistent, whereas non-breeding territorial aggression is transient. Acute inhibition of E2 synthesis significantly inhibits aggressive behavior in male song sparrows in the non-breeding season only (Soma et al., 2000). Further, acute administration of E2 lowers CREB phosphorylation in the medial preoptic nucleus, a brain area implicated in aggression in songbirds, in the non-breeding season only (Heimovics et al., 2012b). These data, taken together with the Peromyscus studies, raise the hypothesis that non-breeding territorial aggression in male song sparrows is transient because it is activated by non-genomic E2 signaling mechanisms. We test this hypothesis here using a non-invasive route of E2 administration to compare the rapid (within 20 min) non-genomic effects of E2 on aggressive behavior in captive male song sparrows during the non-breeding versus breeding season.

Materials and methods Subjects and housing In the Pacific Northwest, song sparrows do not migrate, and males defend territories throughout the year (Arcese, 1989; Wingfield and Monk, 1992). Thus, conspecific song playback and mist nets were used to capture free-living adult male song sparrows both in late October/early November (non-breeding season) and in May (breeding season) near Vancouver, British Columbia (49° 12′N, 123° 01′W). After capture, subjects were transported to the University of British Columbia's Animal Care Centre Annex and housed outdoors in individual wire cages (91 cm × 47 cm × 47 cm) where they were exposed to the natural photoperiod (non-breeding season ≈ 10 L:14D; breeding season ≈ 17 L:7D) and temperature (non-breeding season x ≈ 3. 8 °C; breeding season x ≈ 15 °C). Each cage contained two wooden perches and conifer branches. Seed and water were provided ad libitum, and one wax moth larva was provided daily. Except for behavioral tests, subjects were visually isolated from one another throughout the study. Subjects habituated to captivity for 4–6 weeks prior to the onset of behavioral testing. Breeding season males were in reproductive condition throughout behavioral testing as evidenced by the fact that their gonads were recrudesced and plasma T levels were elevated upon euthanasia one month after the present study was complete (Heimovics et al. in prep.). Similarly, non-breeding season males were in non-breeding condition throughout behavioral testing as evidenced by the fact that their gonads were regressed and plasma T levels were basal upon euthanasia one month after the present study was complete (Heimovics et al. in prep). Protocols were approved by the University

of British Columbia Animal Care Committee and complied with the guidelines of the Canadian Council on Animal Care. Non-invasive route of E2 administration To eliminate stress associated with an injection, we used a noninvasive route of E2 administration. Specifically, E2 (or vehicle control [CON]) was injected into wax moth larvae that were subsequently fed to subjects immediately prior to behavioral testing. Orally administered steroid hormones enter into the blood stream very rapidly (Breuner et al., 1998), and this route of administration has been used previously to examine rapid effects of steroid hormones on behavior in birds, fish, and mice (Breuner et al., 1998; Hodgson et al., 2008; Laredo et al., 2013; Remage-Healey and Bass, 2006; Saldanha et al., 2000). We found that if wax moth larvae are provided to song sparrows daily throughout habituation to captivity, they will consume larva within 30 sec of it being placed in their cage. Thus, this provides a consistent, efficient, and reliable route of steroid administration. The night before each day of testing, larvae were placed in a 4 °C refrigerator in order to render them immobile and cause them to desiccate slightly. The next morning, a Hamilton syringe was used to inject larva with 20 μL of either E2 or CON solution (for details, see below). The syringe was inserted into the ventral surface of the larva, posterior to the last pair of legs. Solution was injected and then the syringe needle was removed slowly to prevent fluid from escaping the larva. In the event that fluid did escape, the larva was discarded and another prepared. Larvae were prepared no more than 5 min prior to being fed to subjects. Preliminary studies Oral E2 dose We previously examined seasonal plasticity in the rapid effects of E2 on the male song sparrow brain (Heimovics et al., 2012b). 15 min after an s. c. injection of 500 μg/kg E2 (12. 5 μg E2 per subject), widespread effects on phosphorylated extracellular signal-regulated kinase (pERK), tyrosine hydroxylase (pTH), and cAMP response element binding protein (pCREB) were observed (Heimovics et al., 2012b). A s. c. dose of 500 μg/kg E2 was selected because this dose, but not lower doses, rapidly modulates sexual behavior in quail (Cornil et al., 2006). Thus, the goal of this preliminary study was to determine an oral dose of E2 that would achieve a similar circulating level of E2 as 12.5 μg s. c. (8. 3 ± 0. 9 ng/mL) (Heimovics et al., 2012b). Wax moth larvae were injected with either 100, 200, or 400 μg E2 (Steraloids) dissolved in (2-hydroxylpropyl)-β-cyclodextrin (0. 5 mg/mL in PBS) (Sigma #C0926). These larvae were subsequently fed to five male song sparrows in a randomized, counter-balanced order with at least 72 h washout between doses. 15 min after ingestion, subjects were captured and blood from the brachial vein was collected into microhematocrit tubes. Tubes were stored on wet ice until centrifugation (within 3 h). After centrifugation, plasma was collected and stored at − 20 °C. E2 levels were measured in plasma in duplicate using a commercially available 125I-E2 radioimmunoassay kit (DSL4800, Ultra-sensitive Estradiol RIA, Beckman Coulter). Methods for this E2 RIA have been published extensively elsewhere (Charlier et al., 2010, 2011; Heimovics et al., 2012b; Taves et al., 2010). Mean plasma E2 levels for each dose was determined, a line of best fit was calculated (y = 1. 1962 + 0. 0274x), and it was concluded that an oral dose of 300 μg E2 would achieve circulating levels of E2 comparable to 12.5 μg E2 s. c. at 15 min after E2 administration (Fig. 1). Importantly, the plasma concentration of E2 achieved here far exceeds plasma levels seen in free-living birds (Soma and Wingfield, 1999b, 2001). However, prior work in songbirds demonstrates E2 levels in brain punches can be two orders of magnitude higher than in plasma (Charlier et al., 2010, 2011; Taves et al., 2011) and E2 concentrations at aromatase-positive synapses may be even higher than in punches

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Plasma E 2 (ng/mL)

15

y 2= 1.1962+0.0274 * x R = 0.9107

10

5

0

12.5

s.c. E 2dose (µg)

100

200

300

400

Oral E2 dose (µg)

Fig. 1. Bar graph (mean ± SEM) representing plasma E2 levels in male song sparrows at 15 min after s.c. injection with 12.5 μg E2 (500 μg/kg) (adapted from (Heimovics et al., 2012b)). Scatterplot and line of best fit illustrating plasma E2 levels in male song sparrows 15 min after eating a wax moth larva injected with 100, 200, or 400 μg E2. The dashed line illustrates the predicted plasma E2 levels in male song sparrows 15 min after ingesting a wax moth larva injected with 300 μg E2 (the dose of E2 used in the present study).

(Saldanha et al., 2011). Additionally, in vitro studies of the rapid, non-genomic effects of E2 typically use doses in the high nanomolar to low micromolar range (which also exceed levels seen in circulation) (Micevych et al., 2007; Pradhan et al., 2008; Woolley, 2007). Oral E2 clearance Previous studies of free-living and captive male song sparrows demonstrate that plasma E2 levels are generally non-detectable in both the breeding and non-breeding seasons (Heimovics et al., 2012a, b; Soma et al., 2004; Soma and Wingfield, 1999a). Given that the present study employs a within-subjects design to transiently increase E2, the goal of this preliminary study was to demonstrate that a 48 h washout period was sufficient for plasma E2 levels to return to baseline (non-detectable) levels. A separate group of four male song sparrows were fed wax moth larva injected with 300 μg E2 and blood from the brachial vein was collected into microhematocrit tubes 1, 24, and 48 h later. To determine baseline plasma E2 levels, subjects were fed a wax moth larva injected with (2-hydroxylpropyl)-β-cyclodextrin (CON; 0. 5 mg/mL in PBS; Sigma #C0926) and blood was collected b 30 sec later. Only one time-point was tested per week per subject and time-points were tested in a randomized, counter-balanced order across subjects. Microhematocrit tubes were handled as above, and E2 levels in plasma were measured via RIA. Note that these subjects showed some evidence of photorefractoriness (molting primary feathers and rectrices). Given this and the time of year (mid-August), it is probable that their gonads were regressing. Results from the RIA indicated that plasma E2 levels at 30 sec and at 24 h and 48 h after ingesting a wax moth larva injected with 300 μg E2 were all below the detection limit of the assay (b 0.2 pg E2 per tube). Plasma E2 levels at 1 h after ingesting an E2-injected larva were 0. 5 ± 0. 3 ng/mL, which is much lower than those seen at 15 min in the “oral E2 dose” preliminary study (Fig. 1). Taken together, these preliminary studies demonstrate that a) non-invasive oral administration of 300 μg E2 (via wax moth larva) leads to a rapid (within 15 min) elevation in circulating E2 levels and b) 48 h is a sufficient washout period for orally-administered E2. Subject pre-screening Several previous studies have utilized a laboratory simulated territorial intrusion (L-STI) to quantify aggressive behavior in captive male song sparrows (Goodson et al., 2005; Sperry et al., 2003, 2010; Wacker et al., 2008). In these prior studies, a cage containing a stimulus (decoy) male and a speaker broadcasting conspecific song were placed

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adjacent to a subject in its home cage for 10–45 min, and then a variety of methods were used to quantify aggressive behavior during the L-STI (Goodson, 2005; Sperry et al., 2003; Wacker et al., 2008, 2010). In the study outlined below, we chose to focus on proximity time and barrier contacts (see below) because these two behaviors are unambiguous, easy to operationally define, and correlate with neural activity in brain regions implicated in aggressive behavior (Goodson et al., 2005). When we utilized the L-STI paradigm described above (including conspecific song playback), only 20% of our randomly assigned “residents” exhibited aggressive behavior. This was most likely because in 41% of preliminary trials, our randomly assigned “decoys” behaved aggressively and dominated social interactions during the social encounter. Further, in these trials, we observed that conspecific song playback was 1) not necessary for birds to exhibit high levels of aggressive behavior and 2) made it more difficult to quantify vocal behavior from videos. For these reasons, in the present study, birds were pre-screened for social dominance during repeated social encounters prior to becoming subjects in the present study and conspecific song playback was not used during L-STI. For pre-screening, male song sparrows (n = 28 non-breeding season, n = 24 breeding season) were randomly assigned to dyads. Dyads were moved in their home cages from outdoor colony housing to an outdoor behavioral testing pen (2 × 3 m) where they remained visually isolated from each other. On the next 3 consecutive days, an opaque partition separating dyad cages was removed and birds were allowed to freely interact through adjacent cage walls (note that no conspecific song playback was employed). 30 min later, the partition was replaced. These 30 min L-STIs were recorded using a Canon Vixia AF 20 HD camcorder placed on a tripod ~ 1 m away from dyad cages. The camcorder's zoom was adjusted so that both cages were completely visible in the view-finder, and a tie clip microphone was attached between the two cages. All dyadic encounters took place between 0900 and 1100. As has been done previously (Goodson et al., 2005), only aggressive behaviors that can be unambiguously quantified (barrier contacts: the number of instances that a bird made full contact (both feet) with the wire barrier separating the two cages and proximity time: the number of minutes that a bird spent in the third of its cage that was adjacent to the wire barrier separating the two cages) were scored from videos. Social dominance was operationally defined as the member of each dyad that made more barrier contacts and had higher proximity time than the other member of the dyad. Remarkably, in all cases but two, social dominance was clear within the first 5 min of the first dyadic encounter and remained stable across all 3 days of testing. Dominant individuals within each dyad became subjects and subordinate individuals became decoys in the experiment outlined below. In the two cases where social dominance was not stable across pre-screening days, neither member of the dyad was used in the experiment outlined below. Additionally, one member of a non-breeding season dyad did not engage in social interactions on any of the days of pre-screening and neither member of that dyad was used in the experiment outlined below. Rapid effect of E2 on aggressive behavior A previous study from this lab investigated seasonal plasticity in the non-genomic effects of E2 on intracellular signaling cascades in male song sparrow brain (Heimovics et al., 2012b). There, subjects were administered fadrozole hydrochloride (FAD, an aromatase inhibitor) via osmotic mini-pump for one week prior to E2 or CON administration. We elected to use FAD in that study because our aim was to examine acute effects of E2 on intracellular signaling cascades in the brain, and we wanted to maximize our ability to detect potentially subtle effects on protein phosphorylation. In the present study, our aim was to examine the acute effects of E2 on a complex social behavior. We sought to minimize stress associated with captivity, handling, and behavioral

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Statistics Data were analyzed using Statistica software (version 9. 1, StatSoft, Tulsa, OK). Data were log transformed prior to analysis when appropriate. To examine seasonal plasticity in the rapid effect of E2 on aggressive behavior, a mixed-design two-factor ANOVA (where season was the between-subjects factor and treatment was the within-subjects factor) was conducted on the number of barrier contacts and proximity time during L-STI. Fisher's LSD post-hoc comparison was used when significant interactions were observed. Note that a mixed-design threefactor ANOVA (where order of treatment was a between-subjects factor) was also conducted to determine whether treatment order (E2 or CON administered first) influenced the outcome of our analyses. We observed neither a significant main effect of order nor a significant season × order, treatment × order, or season × treatment × order interaction effect (p N 0.33 in all cases). Thus only the two-factor ANOVA results are presented below. All figures represent the mean and standard error of the mean for each treatment. p-values ≤0. 05 were considered significant. Eta-squared (η2) and Cohen's d were used to calculate effect size estimates when significant effects were observed. Results The mean (± standard error) latency to consume larva was 6. 8 ± 0. 1 sec (min = 0 sec, max = 22 sec). Latency to consume larva did not differ significantly between seasons or treatments. Rapid effect of E2 on behavior during L-STI The ANOVA yielded a significant season × treatment interaction effect on the number of barrier contacts made during L-STI (F(1,21) = 6. 55, p = 0. 02, η2 = 0.24). Post-hoc comparison revealed a significant difference in the number of barrier contacts made during the L-STI in non-breeding subjects only (Fig. 3A). Non-breeding males made significantly more barrier contacts during L-STI after ingesting an E2-injected larva as compared to a CON-injected larva (p = 0.008, Cohen's d = 0.82). In contrast, E2 had no significant effect on barrier contacts during L-STI in breeding males (p = 0.46). There was no rapid effect of E2 on the number of contacts made with the opposite wall, indicating that the effect of E2 on non-breeding behavior was specific to highly aggressive behavior and E2 did not affect locomotion in general (Fig. 4). The ANOVA also yielded a significant main effect of season on proximity time during the L-STI (Fig. 3B: non-breeding b breeding; F(1,21) = 7. 75, p = 0. 01, η2 = 0.27). Spontaneous song was heard throughout the behavioral testing facility during the breeding season. But, as has been seen previously (Goodson et al., 2005), not enough subjects sang or vocalized during L-STI in this study for statistical analysis.

Subject observed consuming larva During L-STI 10

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ed nd

la

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testing (e.g., we utilized a non-invasive route of E2 administration). Thus, we elected not to pre-treat subjects with FAD because osmotic mini-pump surgery is stressful and also requires additional handling of subjects to verify mini-pumps remain in place throughout behavioral testing. A mixed-design two-factor ANOVA (where season was the between-subjects factor and treatment was the within-subjects factor) was used to test the hypothesis that there are seasonal differences in the rapid effect of E2 on aggressive behavior in male song sparrows. Specifically, two cohorts of birds (non-breeding cohort n = 12, breeding cohort n = 11) were administered both E2 and CON 10 min prior to L-STI. Treatment order (E2 or CON administered first) was randomly assigned to subjects and counterbalanced across subjects/seasons. Behavioral testing for this experiment began on the day immediately following the last day of pre-screening, and there was a 48 h washout period between treatments. In the morning on each day of behavioral testing, a cage containing a decoy novel to the subject was placed immediately adjacent to the subject's cage. The subject cage and decoy cage were separated by an opaque partition. The subject and decoy were then left undisturbed for at least 1 h. Three minutes before the onset of behavioral testing, a Canon Vixia AF 20 HD camcorder was placed on a tripod ~ 1 m away from the subject's cage (Fig. 2). The camcorder's zoom was adjusted so that the subject's entire cage and half of the decoy's cage was visible in the view finder, and a tie clip microphone was attached to the subject's cage. At the onset of trial (T0), video recording began. Then an injected larva (in a small petri dish) was placed on the floor of the subject's cage and the subject and decoy were left undisturbed for 10 min. The order of treatment (300 μg E2 (Sigma #E4389) or CON (2-hydroxylpropyl)-β-cyclodextrin (Sigma #C0926) was counterbalanced across subjects. At T10, the L-STI began (Fig. 2). An experimenter quietly entered the behavioral testing pen, removed the opaque partition separating the subject and decoy cages, and immediately exited the pen. The subject and decoy were then left to freely interact through adjacent cage walls (note: no conspecific song playback was employed). Ten minutes later (T20), the L-STI was terminated (Fig. 2). An experimenter quietly entered the behavioral testing pen, replaced the opaque partition separating the subject and decoy cages, and immediately exited the pen. Ten minutes later (T30) the trial ended (Fig. 2). Subject latency to consume larva and the number of barrier contacts and proximity time during (T10–T20) and after (T20–T30) the L-STI were scored from videos by an experimenter blind to treatment. In addition, occurrences of vocal behavior (number of songs, number of growls) during and after the L-STI were noted. Also, the number of times a subject made full contact (both feet) with the wire barrier opposite the decoy's cage during the L-STI was scored from videos and served as a behavioral control.

Tr ia

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Fig. 2. Schematic diagram of the behavioral testing protocol. Numbers reflect time (in min) each event occurred. Arrow represents the maximum latency to consume injected larva observed in the study (22 sec). Brackets represent the 10 min time periods operationally defined as “during” and “after” the laboratory simulated territorial intrusion (L-STI).

S.A. Heimovics et al. / Hormones and Behavior 69 (2015) 31–38

Number of barrier contacts

A

B 45 40 35 30 25 20 15 10 5 0

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Aggressive behavior during L-STI Fig. 3. Bar graphs (mean ± SEM) representing (A) the significant season × treatment interaction effect on the number of barrier contacts and (B) the significant main effect of season on proximity time observed during the L-STI. * p ≤ 0. 05.

Rapid effect of E2 on behavior after L-STI Notably, the effect of E2 on barrier contacts was specific to the period during the L-STI. That is, there was no main effect of season, main effect of treatment, or a season × treatment interaction effect on barrier contacts after the L-STI (Fig. 5A). However, the ANOVA indicated a significant main effect of season on proximity time after L-STI (Fig. 5B non-breeding b breeding; F(1, 21) = 9. 27, p = 0. 006, η2 = 0.31). No songs or other vocalizations were observed after the L-STI. Discussion This study tested the hypothesis that non-breeding (but not breeding) season territorial aggression in male song sparrows is activated by non-genomic E2 signaling mechanisms. Consistent with this hypothesis, acute, non-invasive E2 administration significantly increased the number of barrier contacts made during a 10 min L-STI in non-breeding season subjects only. Because the effect of E2 on barrier contacts was seen less than 20 min after administration, it is likely that E2 acted via non-genomic mechanisms (Cornil and Charlier, 2010). To our knowledge, this is the first time a non-invasive route of steroid administration has been used to alter a complex social behavior in a songbird. Taken together with the natural history of the song sparrow, this study provides novel insight into the adaptive value of rapid, transient, non-genomic mechanisms of steroid action.

aggression were observed 10–20 min after E2 administration; a time course that is incompatible with a classic genomic model of steroid hormone action (Cornil and Charlier, 2010). Thus, it is likely E2 increased aggression via a non-genomic signaling mechanism. In this study, E2 had no rapid effect on breeding season aggression. Thus, there appears to be seasonal plasticity in the non-genomic effects of E2 on agonistic behavior in male song sparrows. Several lines of evidence support this conclusion. First, research in Peromyscus mice shows that E2 rapidly alters aggressive behavior in animals housed under short-day, but not long-day, photoperiods (Trainor et al., 2007a, 2008). Second, prior work in free-living song sparrows shows that acute inhibition of E2 synthesis lowers aggression during the non-breeding, but not the breeding, season (Soma et al., 2000). Third, STI in non-breeding song sparrows rapidly increases the local production of aromatizable androgens within brain areas implicated in aggression (Pradhan et al., 2010). This is important because it has been hypothesized that steroids produced locally in the brain (i. e. neurosteroids) are more likely to act via non-genomic mechanisms than systemic steroids (Schmidt et al., 2008; Woolley, 2007). Fourth, in white-crowned sparrows, the rapid behavioral effects of corticosterone also vary with photoperiod (Breuner and Wingfield, 2000). Thus, the present data taken together with past work suggest that environmental cues associated with the non-breeding season (photoperiod, temperature) influence the molecular mechanisms through which steroids influence behavior. Future studies should examine the rapid effects of membrane-impermeable E2 and/or of E2 paired with a protein synthesis inhibitor.

Seasonal plasticity in the non-genomic effects of E2 on aggression

Proximate mechanism for a rapid effect of E2 on non-breeding aggression

Here, E2 rapidly increased barrier contacts made during L-STI in non-breeding season male song sparrows only. The effects of E2 on

Multiple intracellular signaling cascades are associated with nongenomic E2 signaling. E2 rapidly modulates calcium flux (Mermelstein et al., 1996), activates metabotropic glutamate receptors (Meitzen and Mermelstein, 2011), and alters kinase activity including extracellular signal-regulated kinase (ERK) (Singer et al., 1999), among others (Ivanova et al., 2002; Kelly and Wagner, 1999). Any of these intracellular signals could have mediated the effect of E2 on non-breeding aggression observed in this study. A study that examined the rapid effect of E2 on ERK phosphorylation and its targets (cAMP response element binding protein (CREB) and tyrosine hydroxylase (TH)) provides insight into brain regions that may have mediated the behavioral effects observed here. E2 rapidly (within 15 min) alters phosphorylated CREB (pCREB) levels in the medial preoptic nucleus (POM) in non-breeding (but not breeding) condition male song sparrows (Heimovics et al., 2012b). Estrogen synthesis within the preoptic area has been implicated in aggressive behavior in birds (Ubuka and Tsutsui, 2014), and recent work in white-throated sparrows highlights the POM as central to ERα-mediated control of territorial aggression in songbirds (Horton et al., 2014). ERα is typically found in the cytosol, but it can also be

Number of contacts with opposite wall

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Non breeding

Locomotor behavior during L-STI Fig. 4. Bar graphs (mean ± SEM) representing no effect of season, treatment or season × treatment on the number of contacts made with the opposite wall during the L-STI.

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B

Number of barrier contacts

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**

500

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400 300 200 100

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Non breeding

Aggressive behavior after L-STI Fig. 5. Bar graphs (mean ± SEM) representing (A) no effect of season, treatment, or season x treatment on the number of barrier contacts after the L-STI and (B) the significant main effect of season on proximity time after the L-STI. ** p ≤ 0. 01.

trafficked to the plasma membrane (Micevych and Dominguez, 2009). ERβ is also implicated in vertebrate aggression (Nomura et al., 2002, 2006; Trainor et al., 2007b), is expressed in the POM of songbirds (Bernard et al., 1999), and can also be associated with cell membranes (Razandi et al., 1999). Additionally, GPER1 (a.k.a. GPR30, a membranebound G protein-coupled receptor with high affinity for E2 (Kelly and Ronnekleiv, 2008; Toran-Allerand, 2005)), is expressed in the songbird brain (Acharya and Veney, 2012) and recent studies show dense immunoreactivity (primarily beaded fibers) for GPER1 within POM of male song sparrows (Soma et al., unpublished results). Taken together, it is plausible that membrane-associated ERα, ERβ or GPER1 within the POM mediated the rapid effect of E2 on aggression observed here. Rapid effects of E2 on pCREB, pERK, and pTH in nucleus taeniae of the amygdala (TnA), ventromedial hypothalamus (VMH) and mesencephalic central gray (GCt) of male song sparrows have also been observed (Heimovics et al., 2012b). TnA, VMH, and GCt are part of the vertebrate social behavior network and regulate multiple forms of social behavior, including aggression (Goodson, 2005). ERα mRNA is expressed in TnA and VMH (Horton et al., 2014; Rosvall et al., 2012), and mRNA levels in TnA are positively associated with aggressive behavior in dark-eyed juncos (Rosvall et al., 2012). ERβ mRNA is expressed in the songbird hypothalamus (Bernard et al., 1999), and neither ERα nor ERβ mRNA levels vary seasonally in male song sparrow brain (Wacker et al., 2010). GPER1-ir has also been detected in VMH and GCt of male song sparrows (Soma et al., unpublished results). Thus TnA, VMH, and GCt are also possible candidate brain regions for mediating the effects of E2 on behavior observed here. Future studies utilizing site-specific administration of E2 to POM, TnA, VMH, and GCt would provide insight into the relative contribution of each region to the neuroendocrine control of aggression. Importantly, membrane-associated corticosteroid receptor number varies seasonally in house sparrow brain (Breuner and Orchinik, 2001). Seasonal variation in membrane associated ER levels would provide insight into why non-breeding subjects appear to be more sensitive to exogenous administration of E2. Alternatively, seasonal variation in ER affinity might also explain why a rapid effect of E2 was only observed in non-breeding subjects. Both of these possibilities should be explored in future research. Adaptive value of non-genomic activation of behavior In many species, territorial aggression is limited to the breeding season and functions to obtain and maintain access to mates. In species that are territorial year-round, the function of aggressive behavior in the non-breeding season is different. Many of these species do not migrate (e. g. Pacific Northwest song sparrows (Arcese, 1989; Wingfield and Monk, 1992), red squirrels (Boonstra et al., 2008)) and there is high metabolic demand associated with over-wintering at high latitudes (Rogers, 1995; Rogers et al., 1991). Thus, the function of non-breeding

territoriality is to protect food resources essential to over-winter survival. This seasonal difference in the function of aggression parallels seasonal differences in the persistence of aggression seen in song sparrow field studies. In field studies, after a STI ends, breeding males remain aggressive for hours (even days) while non-breeding males rapidly stop behaving aggressively within minutes (Wingfield, 1994). Transient aggression in the non-breeding season is adaptive: it minimizes energy expenditure and maximizes the amount of time available for foraging (which is already limited because days are shorter) (Wingfield, 1994). The natural history of the song sparrow taken together with the present data suggests that territorial aggression during the non-breeding season may be activated by a non-genomic mechanism due to the fitness benefits associated with rapid and transient expression of aggressive behavior. During the non-breeding season, gonads are regressed and circulating T is non-detectable (Wingfield and Hahn, 1994). When levels of aromatizable androgens are low in circulation, data suggest that the brain synthesizes E2 either de novo from cholesterol or from dehydroepiandrosterone (Soma et al., 2008). Neurosteroids are more likely to act via non-genomic mechanisms than peripherally produced steroids (Schmidt et al., 2008; Woolley, 2007) Thus, a shift to non-genomic activation of behavior in the non-breeding season may also have evolved to avoid the costs associated with steroids being chronically elevated in the blood (e. g. decreased body mass, suppressed immune function, etc.). There are substantial fitness costs associated with unsuccessful territory defense during the breeding season (e. g. loss of breeding territory, cuckoldry). Given these costs, it is not surprising that we observed no rapid, non-genomic effect of E2 on breeding season aggression because transient expression of aggressive behavior in this context would be maladaptive. Our prior work suggests that breeding and non-breeding song sparrows do not differ in peripheral metabolism of exogenous E2 (Heimovics et al., 2012b). However, seasonal differences in central metabolism/catabolism of E2 have not been systematically evaluated and thus could provide an alternative explanation for the lack of a rapid effect of E2 on breeding season aggression. This possibility should be explored in the future. Note that in the present study we saw no evidence of seasonal plasticity in the persistence of aggression seen in field studies. This is probably because the “persistent behaviors” quantified in field studies (spontaneous singing, patrolling of a large territory) (Wingfield, 1994) are difficult to observe/quantify using an L-STI paradigm. Also, in field studies decoy cages are typically removed after the STI whereas in our L-STI paradigm we only placed an opaque partition between the subject and decoy cages. But, in the present study, we did observe that proximity time during and after L-STI is significantly higher in breeding males than non-breeding males. While this is not a direct measure of persistence, it may indicate that viewing an intruder induces a higher

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state of arousal, stress, and/or anxiety-like behavior in breeding condition subjects. Consistent with this idea, pTH in the lateral septum (LS) is higher in breeding than in non-breeding season males (Heimovics et al., 2012b). TH is the rate-limiting enzyme in catecholamine synthesis, and the release of norepinephrine within the LS is plays an important role in the neuroendocrine regulation of arousal (Morilak et al., 2005). The role of catecholamines in the regulation of persistent aggression in the breeding should be explored with future research.

Conclusions Animals living at temperate latitudes experience profound seasonal changes in physiology and behavior, but it is not uncommon for behaviors such as territorial aggression to be observed throughout the year. However, the function of and underlying motivational state accompanying these behaviors differ seasonally, and the present data taken with past work shows that the proximate mechanisms regulating these behaviors can differ seasonally as well. Non-genomic activation of E2-dependent behavior appears to predominate during the nonbreeding season, and this study highlights one ecological context in which a rapid, transient mechanism of hormone action is highly adaptive.

Acknowledgments We thank Nora Prior and Annika Sun for help with field work, Tal Kaikov for help with behavioral testing, the University of British Columbia Animal Care Centre Annex staff for help with animal care, Dr. Cathy Ma for assistance with the E2 assays, and Dr. Joanne Weinberg for the use of her gamma counter. This work was supported by postdoctoral fellowships to S. A. H. from the Canadian Institutes of Health Research (CIHR) and the Michael Smith Foundation for Heath Research and a CIHR Operating Grant to K. K. S. (#67087).

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