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Coordinated and dissociated effects of testosterone on singing behavior and song control nuclei in canaries (Serinus canaria)
Hormones and Behavior 47 (2005) 467 – 476 www.elsevier.com/locate/yhbeh
Coordinated and dissociated effects of testosterone on singing behavior and song control nuclei in canaries (Serinus canaria) Jennifer J. Sartora,*, Jacques Balthazartb, Gregory F. Balla a
Department of Psychological and Brain Sciences, Ames Hall, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA b Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology, University of Lie`ge, 17 Place Delcour (Bat. L1), B-4020 Lie`ge, Belgium Received 28 September 2004; revised 1 December 2004; accepted 3 December 2004
Abstract Temperate zone songbirds that breed seasonally exhibit pronounced differences in reproductive behaviors including song inside and outside the breeding season. Springlike long daylengths are associated with increases in plasma testosterone (T) concentrations, as well as with increases in singing and in the volume of several brain nuclei known to control this behavior. The mechanisms whereby T can induce changes in behavior and brain, and whether or not these effects are differentially regulated, have recently begun to be examined, as has the question of the relative contributions of T and its androgenic and estrogenic metabolites to the regulation of this seasonal behavioral and neural plasticity. In this experiment, we examined the effects of T, 5a-dihydrotestosterone, or 17h-estradiol treatment on castrated male canaries housed on short days and compared neural and behavioral effects in these males to similarly-housed males given only blank implants. We observed that only T treatment was effective in eliciting significant increases in singing behavior after 11 days of hormone exposure. In addition, T alone was effective in increasing the volume of a key song production nucleus, HVC. However, at this time, none of the steroids had any effects on the volumes of two other song control nuclei, Area X of the medial striatum and the robust nucleus of the arcopallium (RA), that are efferent targets of HVC, known to be regulated by androgen in canaries and also to play a role in the control of adult song. T can thus enhance singing well before concomitant androgen-induced changes in the song control system are complete. D 2004 Elsevier Inc. All rights reserved. Keywords: Songbird; Birdsong; Neural plasticity; Seasonal; Estrogen; DHT; Area X; RA; Activity-dependent plasticity
Introduction In temperate zone birds, reproductive behaviors follow an annual cycle consisting of periods of breeding and nonbreeding. Courtship and reproductive behaviors thus occur in most species in the spring, when temperatures are relatively mild and food availability is highest (reviewed in Wingfield and Kenagy, 1991). Changes in photoperiod provide a valuable cue from the environment that allows animals to assess the time of year. The ability to detect and respond to photoperiodic changes has evolved in many avian species living in either the tropics or the temperate
* Corresponding author. Fax: +1 410 516 4478. E-mail address: [email protected] (J.J. Sartor). 0018-506X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2004.12.004
zone (Dawson et al., 2001; Murton and Westwood, 1977; Nicholls et al., 1988; Wilson and Donham, 1988). Physiological responses to photoperiod are mediated by a biological system that includes both a neural as well as an endocrine component (Follett, 1984), which is characterized by seasonal changes in the size of the gonads as well as the blood plasma levels of sex steroid hormones such as testosterone (T; reviewed in Ball and Balthazart, 2002). In addition, springtime increases in testis volume and plasma T concentration are associated with increases in courtship behaviors, of which song is a major component in males of many species of temperate-zone, seasonally-breeding songbirds (see Ball et al., 2004 for review). Male canaries (Serinus canaria), like many temperate zone oscines, exhibit seasonal changes in the volumes of many song control nuclei involved in song production such
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as HVC and the robust nucleus of the arcopallium (RA; for a discussion of the new avian telencephalic nomenclature see Reiner et al., 2004) as well as in Area X of the medial striatum, which is involved in song learning and the contextual production of song (Nottebohm, 1981; reviewed in Ball, 1999; Schlinger and Brenowitz, 2002; although see Leitner et al., 2001). Testosterone is a major mediator of these variations. Laboratory studies of canaries have demonstrated that volumes of the song control nuclei are large when T concentrations in the blood are high, and these nuclei are smaller when T concentrations are decreased (Nottebohm et al., 1986, 1987). Testosterone can be metabolized into the non-aromatizable androgen 5a-dihydrotestosterone (DHT) by the enzyme 5a-reductase, or into estradiol (E2) by the enzyme aromatase (ARO; see Ball and Balthazart, 2002; Harding, 1986 for reviews). Both androgen (Arnold et al., 1976; Balthazart et al., 1992; Bernard et al., 1999; Smith et al., 1996; Soma et al., 1999) and estrogen (Bernard et al., 1999; Gahr et al., 1993) receptors are located within specific song control nuclei such as HVC in canaries and other species. Furthermore, HVC is also surrounded by high expression and activity of ARO (Balthazart et al., 1996; Shen et al., 1995), implying that not only androgens but also estrogens could have important actions in this nucleus. The song production nucleus RA is a target for androgens but contains no receptors for estrogenic metabolites (Balthazart et al., 1992; Bernard et al., 1999). Given this distribution of steroid hormone receptors and metabolizing enzymes in the songbird telencephalon and song control nuclei, it is possible that the mediation of seasonal volumetric changes by T could be dependent on one of its metabolites, or could result from a synergistic action of T and its metabolites at the various sites. In ovariectomized female canaries treated with either T, DHT, or E2, singing was only observed in T-treated females, and dendritic branching in RA was longer in T-treated females than in females from any other hormone treatment group (DeVoogd and Nottebohm, 1981), implying that androgenic and estrogenic metabolites on their own cannot induce these changes, but that these changes may require synergistic effects. In other species of songbirds, it has in fact been shown that both singing as well as changes in morphology of song control nuclei is dependent on the synergistic actions of androgens and estrogens. Castrated male zebra finches (Taeniopygia guttata; Harding et al., 1983; Walters and Harding, 1988) and red-winged blackbirds (Agelaius pheoniceus; Harding et al., 1988) do not engage in sexual behaviors and restoration of these behaviors requires treatment with both androgens and estrogens to restore the full complement including song. In Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii), however, treatment of castrated males with either androgens or estrogens, or a combination of both, was sufficient to elevate the volumes of HVC, RA, and Area X above those for controls without significantly elevating singing behavior (Tramontin et al., 2003).
The sequence and time course of T action on brain and singing behavior are still poorly understood. Recently, however, studies in free-living songbird species have indicated that there could be a dissociation between the behavioral and neural actions of T, such that T may not act uniformly throughout the song system to activate growth and singing behavior, but may instead activate some aspects more quickly than others. For example, in male wild-caught Gambel’s white-crowned sparrows, exposure to T and long daylengths was sufficient to activate growth of HVC and to instigate changes in singing behavior by 7 days posttreatment (Tramontin et al., 2000). Area X and RA, however, were not significantly larger than in control males until 20 days after initial exposure, and song stereotypy (important for singing in a reproductive context) was not fully developed until 20 days after T and long-day exposure. These results suggest that the song system, and singing behavior, are both stimulated by T but that this occurs in a sequential manner, with HVC and song output changing on a quicker timeline than Area X and RA (Tramontin et al., 2000). Furthermore, activation of HVC by T is sufficient to stimulate the growth of its efferents RA and Area X sequentially, whether T is administered systemically (Brenowitz and Lent, 2001) or implanted directly into HVC in castrates (Brenowitz and Lent, 2002). One implication of these experiments is that complete recrudescence of song control nuclei is not necessary for increases in song production, indicating a dissociation between the effects of T on behavior and the brain nuclei known to control that behavior. Additional lines of evidence have developed in the past several years that indicate a dissociation between the effects of T on song control nuclei and singing behavior. For instance, European starlings (Sturnus vulgaris) sing outside the breeding season, when plasma T concentrations are low (Eens, 1997; Feare, 1984) and volumes of the song control nuclei are smallest (Riters et al., 2002). In this species, then, song production per se is not tightly correlated to complete development of song control nuclei and can therefore occur without recrudescence of these regions. The volumetric development of song control nuclei may in these cases relate more to changes in song stereotypy or complexity. In this study, we examined the onset of the effects of T or its metabolites on singing and on song control nuclei in male canaries that were castrated, implanted with either T, DHT, or E2, and maintained on short daylengths for 11 days. We decided upon this length of time for exposure to the steroids by using the hormonal induction of the behavior we were interested in, singing, to guide our investigation of the hormonal initiation of growth of the nuclei involved in the production of that behavior. T-treated males began singing 4 days after hormone implantation, and we observed singing behavior for 1 week after that, at which time there was a significantly higher rate of singing in T-treated males compared to any other groups. By housing these males on short days, we were able to examine the effects of steroid
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treatments in the absence of photoperiodic stimulation to discriminate effects due to the steroids from those of the photoperiod per se. Using this procedure, we observed the development of aggressive and song behavior in these canaries for 11 days following steroid hormone implantation and then sacrificed all subjects for the measure of the song control nuclei volumes when song rates had significantly increased in T birds. This experiment demonstrated that T, but not any of its metabolites on their own, acts first to stimulate song behavior and HVC volume but requires longer exposure to induce growth in Area X and RA, even in the absence of stimulating long daylengths. High rates of singing behavior precede RA and Area X growth, indicating that hormonal effects on behavior do not require increased volumes of all song control nuclei, and demonstrating that changes in a behavior are not necessarily preceded by changes in the volumes of several of the nuclei known to be involved in the control of that behavior.
Materials and methods Experimental animals Twenty adult male canaries were obtained from a breeder in Lie`ge, Belgium and housed indoors in plastic cages (49 95 51 cm) in groups of 4 in the laboratory of Dr. Jacques Balthazart at the Universite´ de Lie`ge on an 8 L:16 D (light/ dark) light cycle. After 5 weeks, all males were castrated under general anesthesia induced by injection of 0.1 ml of a mixture containing 4.33 ml saline solution, 0.33 ml xylazine (Rompun; 20 mg/ml), and 2.10 ml ketamine (Ketalar; 50 mg/ ml). All testes were regressed in these males at the time of castration, indicating that males were in a nonbreeding condition. The two testes were removed through a unilateral incision made posterior to the last rib along the left flank. Forceps were placed at the base of each testis and the whole testis was removed from each side in one motion. The incision was then sutured closed and males were allowed to recover before being returned to their home cages. Following recovery, males were housed an additional 8 weeks on 8 L:16 D in groups of four to ensure that they would respond to the hormone treatments when they were administered. After 8 weeks, all males were implanted subcutaneously with one Silastic implant (Dow Corning, Midland, MI, USA, no. 602-175; 0.76 mm inner diameter, 1.65 mm outer diameter) filled to a length of 10 mm with either crystalline T, DHT, or E2, or left empty as control (blank implants). The doses of hormone used (length and diameter of implants) were selected based on previously published literature indicating that T implants of this size are able to activate singing and to increase the sizes of song control nuclei in canaries to levels characteristic of normal, gonadally-intact males (Appeltants et al., 2003; Nottebohm, 1980). We used similar lengths of implants for the androgenic and estrogenic metabolites of T as we did for
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T itself. It should be remembered however that the release of steroids from Silastic tubing varies inversely with polarity, such that more polar steroids such as E2 diffuse much more slowly, resulting in lower doses from the same lengths of tubing (Balthazart and Hirschberg, 1979). In addition, given that central actions of E2 and to some extent DHT as well are largely controlled by the local metabolism of T at the site of action (reviewed in Ball and Balthazart, 2002), the relevant physiological concentration for estrogenic and androgenic metabolites of T that must be considered is not in the plasma but in the brain. There are no data available indicating the local brain concentrations that will be produced by different implant sizes. It is clear however that the sizes selected here for E2 and DHT implants are able and actually required to activate aspects of reproductive behaviors in a variety of avian species (Balthazart et al., 1985; Kreutzer and Vallet, 1991; Leboucher et al., 1994; Schumacher and Balthazart, 1983). In canaries, the length of E2 implants used has been shown to be physiologically relevant and to elevate E2 to concentrations similar to those seen in photostimulated female canaries (Leboucher et al., 1994). One bird from each cage received one of the four types of implants. Thus, there were 5 birds in each treatment group, with one bird from each group living in each of the five cages for behavioral testing. Males were group-housed in this manner so that aggressive and song behavioral testing could be carried out at the same time for all birds. Two males, one blank-implanted and one T-implanted, died during the behavioral observation period. Behavioral testing All males remained on an 8 L:16 D photoperiod for the duration of behavioral testing, so that the effects of steroid hormone treatment could be examined in the absence of any other cues characteristic of breeding conditions, particularly long daylengths. Testing commenced at the time the lights turned on in the animal room each day, beginning on the day following implantation, and continued for 2 h each day for 10 days. In one observation hour, singing behavior was scored. Each cage was observed for a total of 10 continuous minutes and singing was scored every time it occurred for all males. This method of behavioral observation has been previously shown to be effective for capturing rates of these behaviors in canaries (see Appeltants et al., 2003). In the second hour of observation, singing was again recorded in the same fashion. In addition, during this hour, aggressive behaviors, including chasing, pecking, fighting, and the adoption of aggressive postures (in which males would crouch with wings waving and bills open towards other males), were scored throughout the observation period for all birds. Behavioral observations were concluded and brains were collected when high rates of singing had been observed in some males for a period of 7 days. Singing began on Day 4 of testing and males were therefore sacrificed 11 days following hormone treatment. The order
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of observation sessions was counterbalanced across days of testing.
Results Behavioral characterizations
Perfusions All 20 males from the experiment were perfused on the same day, immediately following behavioral data collection. Once behavioral testing was completed, males were sacrificed one cage at a time, in a random order within the cage, and brains were removed and stored for volume reconstruction of the song control nuclei. Presence of an implant was verified in each bird at the time of sacrifice. Prior to perfusion, males were injected with 0.03 ml of heparin (20 mg/ml, i.m.) and deeply anesthetized with 0.2 ml of the anesthetic mixture described above. An additional 0.03 ml of heparin was injected directly into the heart and males were perfused through the left ventricle with approximately 80 ml of 0.9% saline, followed by 200 ml of fixative (4% paraformaldehyde (PAF) in 0.1 M phosphate buffer, pH 7.2). Brains were dissected out of the skull and postfixed overnight in PAF. The brains were transferred into a sucrose solution (30% sucrose in 4% PAF) for an additional two nights. Following this, brains were frozen on dry ice and stored at 708C until processed for volume reconstruction. Frozen brains were cut in five series in coronal sections at 30 Am using a cryostat. All sections were collected into 1ml Eppendorf tubes containing 4% PAF and stored at 48C until processed. The first series was collected for Nissl staining; the remaining four series of sections were set aside and saved for procedures not described here.
To investigate possible differences in singing behavior for males in the different steroid hormone treatments, the data were analyzed using a one-way analysis of variance (ANOVA) comparing song rates in the four groups of males. The results of this analysis revealed a significant difference among the four conditions (F(3, 14) = 23.46; P b 0.0001; Fig. 1A). Fisher’s protected LSD tests revealed that song rates were significantly higher in T-treated males compared to all other groups (P b 0.001 for each comparison). No other differences between groups were detected. Singing behavior began 4 days after hormone treatment, and Fig. 1B shows the increase in singing
Song nuclei volume reconstruction Brain tissue was Nissl-stained using Thionin. The volume of the song control nuclei HVC, RA, and Area X were calculated for each bird using a Zeiss Axioscope microscope with a CCD camera connected to a Macintosh computer. Each of these nuclei is denser in Nissl-stained material than the surrounding tissue and the boundaries can be easily identified. Toward the caudal end of HVC, this nucleus has been subdivided into an binclusiveQ part and an bexclusiveQ part located medially along the lateral ventricle (Kirn et al., 1989). For the purposes of volume reconstruction in this report, we measured only the binclusiveQ HVC. This also means that we did not include para-HVC sensu Johnson and Bottjer (1993). The brain images were digitized and the area of each nucleus was traced on the digitized section using the program NIH Image (Version 1.62, Wayne Rasband, Bethesda, MD; for HVC only), or the program OpenLab (version 2.0, Improvision Inc.). Area measurements were made bilaterally through the rostral to caudal extent of the nucleus by tracing its perimeter. To derive volume estimates, areas were summed and multiplied by the sampling interval, in this case 0.150 mm. No systematic asymmetries were found, and all reported values represent the mean of the two sides for each nucleus.
Fig. 1. Effects of different steroid hormone treatments on singing behaviors in castrated canaries housed on short (8 L:16 D) photoperiods. Error bars represent standard errors in both cases. (A) Only treatment with T produced singing behavior, and this increase with T exposure was significant compared to all other groups. The asterisk indicates a significant difference in singing behavior at P b 0.05. (B) Song rate increased with T treatment within 4 days following implantation, and remained high for the duration of the experiment. No metabolites of T were effective at elevating song rate above castrated values. Bl = blank implant.
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behavior across the test days, which was only observed in the groups treated with T and DHT. All aggressive behaviors were summed for each male for each testing session and analyzed as one measure. Testosterone-treated males were the one group who exhibited any appreciable amount of behavior, but this difference was not statistically significant due to large standard errors in each group (F(3, 14) = 0.93; P N 0.4; Fig. 2). In the males exhibiting aggression, behaviors largely included chasing of other males, with some pecking and fighting, and very little aggressive posturing. Steroid hormone treatment and HVC volume One-way ANOVA revealed a significant difference in HVC volumes among the four treatment groups (F(3, 14) = 4.38; P b 0.03). Fisher’s protected LSD tests indicated that the volume of the song production nucleus HVC was significantly larger in T-implanted male canaries than in all other groups (P b 0.02 for all comparisons; Fig. 3A). No other groups differed from any other. We also used an analysis of covariance (ANCOVA) with HVC volume as the dependent measure, steroid hormone treatment as the categorical variable, and song rate as the continuous predictor to look for effects of the hormone treatments when song rate is presumed to covary. We did not find any significant effects of either hormone implant or song rate on HVC volume using this method. A simple linear regression of song rate (songs/h) onto HVC volume (mm3) revealed that these two variables were significantly correlated (r = 0.61; P b 0.01; Fig. 3B). Steroid hormone treatment and RA volume One-way ANOVA revealed that the volume of the song production nucleus RA was not different among any of the
Fig. 3. Effects of different steroid hormone treatments on the volume of the song production nucleus HVC in castrated male canaries housed on short photoperiods. (A) Only treatment with T was effective in significantly elevating HVC volume above baseline under these conditions. The standard errors of each mean are represented by the error bars, and the asterisk indicates significance at P b 0.05. (B) Song rate (in songs/h) was positively correlated with HVC volume in T-treated male canaries ( P b 0.05).
hormonal treatments (F(3, 14) = 1.03; P N 0.4; Fig. 4A). There was no significant correlation between song rate (songs/hr) and RA volume (mm3) as demonstrated using simple linear regression (r = 0.23; P N 0.3; Fig. 4B). Steroid hormone treatment and Area X volume The volume of nucleus Area X was not different for any of the hormonal conditions when tested using one-way ANOVA (F(3, 14) = 2.77; P N 0.08; Fig. 5A). A simple linear regression of song rate (songs/h) onto Area X volume (mm3) revealed that these two variables were significantly but negatively correlated (r = 0.54; P b 0.03; Fig. 5B). Fig. 2. Effects of different steroid hormone treatments on a variety of aggressive behaviors (see text) in castrated canaries housed on short photoperiods. Although there were no statistically significant differences between the groups, males treated with T tended to show higher rates of aggressive behaviors compared to other treatment groups.
Discussion In seasonally-breeding songbirds, the association between long, springtime daylengths, high levels of blood
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Fig. 4. Effects of different steroid hormone treatments on the volume of the telencephalic song production nucleus RA in castrated male canaries housed on short photoperiods. (A) There was no effect of any of the steroid hormones on the volume of RA. Error bars represent the standard errors of the means. (B) Song rate (in songs/h) was not significantly correlated with RA volume in male canaries.
plasma testosterone, and increases in song rate and the volume of telencephalic nuclei known to control this behavior has long been apparent (see Ball et al., 2004 for review). Specific models for the regulation of seasonal neuroplasticity by T and the mechanism whereby these variables are related have only begun to be investigated. Recent work in wild-caught white-crowned sparrows has indicated that the timeline for the activation of singing behavior by testosterone may differ from that required for T to have effects on the growth of many of the song control nuclei involved in the production of that behavior, which is known to be regulated by T (Tramontin et al., 2000). In castrated males of this species, treatment of males with T and exposure to long days resulted in increases in both HVC size as well as singing behavior within 7 days of treatment administration, whereas increases in the sizes of the efferent targets of HVC, RA, and Area X were not observed until 20
days post-treatment (Tramontin et al., 2000). In the present experiment, we examined volumes of these song control nuclei in castrated male canaries, housed on short days and treated with either T or one of its metabolites, at a time when a full induction of song rate was observed in T-treated subjects. We therefore selected the length of time that these males would be exposed to their hormonal treatments based on the exhibition of the behavior in which we were interested, and then examined the brain nuclei involved in that behavior once singing had been induced. By housing these males on short days throughout the duration of the experiment, we were able to examine the effects of exposure to T and its metabolites on brain and behavior when the only indicator of a bbreeding conditionQ is plasma hormone concentration. Increasing photoperiods may have stimulatory effects on song system growth even in the presence of small gonads and/or low testosterone
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Fig. 5. Effects of different steroid hormone treatments on the volume of the anterior forebrain nucleus Area X in castrated male canaries housed on short photoperiods. (A) There was no effect of any of the steroid hormones on the volume of Area X. Error bars represent the standard errors of the means. (B) Song rate (in songs/h) was negatively correlated with Area X volume in male canaries ( P b 0.05).
concentrations (Bernard et al., 1997; Tramontin et al., 2001). We found further evidence for the model proposed by Tramontin et al. (2000) in white-crowned sparrows and observed that in canaries, short-term T treatment, even in the absence of stimulating daylengths, is sufficient to activate singing behavior and to increase the volume of HVC, but does not have any effect on its targets, RA and Area X. These data demonstrate that T can relatively rapidly activate some aspects of seasonal neural and behavioral plasticity, whereas other aspects require longer exposure times to high titers of this steroid. The lack of effect of any other steroid hormone on volumes of any song control nuclei is in agreement with previous work in hormonetreated canaries, where only T treatment was found to induce singing behavior and the growth of dendritic spines in RA (DeVoogd and Nottebohm, 1981). The effects of T on RA were observed after 4 weeks of T treatment in this case.
Song rate was correlated with HVC size in these birds but was not significantly correlated with RA volume. The correlation between HVC volume and rate of song production agrees with previous work on singing behavior in canaries (Nottebohm et al., 1987). The lack of correlation between singing and RA size is not surprising given that none of the steroid treatments had any effect on RA size. Surprisingly, the volume of Area X was negatively correlated with song rate in these birds. The causal links potentially explaining this negative correlative remain however unclear at present. At the doses used here and under these conditions, treatment of male canaries with only DHT or E2 alone was not sufficient to elicit any significant change in either brain or behavior in this species. It must be noted however that in this experiment we housed the males in groups of 4 with one male from each treatment group in each cage. We chose this experimental design to avoid issues of pseudo-replication
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that would have occurred had we housed birds from the same hormonal condition together. By using this design, we might have established a situation where T-treated males, because they were active singers and more aggressive, may have suppressed these behaviors in the DHT- and E2-treated males with which they were housed. This suppression of behavior could have led to a subsequent lack of growth in song control nuclei in these males and may explain the lack of effect of the T metabolites on song control nuclei growth. It cannot explain, however, the observation that T acts more quickly on behavior and on HVC volume than on other telencephalic song control nuclei. It should be noted however that these housing conditions may have affected the expressions of aggressive behaviors in groups that did not receive T treatment. Another possible explanation for the lack of effect of any hormones other than T could concern the doses used in this experiment. This seems unlikely, however, given that we chose these doses based on previous literature where similar or shorter lengths of implants were shown to elevate blood plasma concentrations of steroid to physiological levels (for example, see Brown et al., 1993; Nottebohm, 1980). It is thus more likely that neither DHT nor E2 treatment alone significantly changed brain and behavior either because of the limited length of time during which animals were exposed to the hormones, or because synergistic actions between estrogenic and androgenic metabolites are required (see below). In addition, it is possible that the process of song activation and/or seasonal increases in HVC volume requires the metabolization of T into either E2, DHT, or both, but that this must occur at the site of action of these hormones to attain an effective concentration of the hormone locally. This would potentially have occurred with the T treatment described here, and could indicate that a synergy between androgenic and estrogenic metabolites of T is necessary to produce changes in both brain and behavior. This would be one way of achieving high local concentrations of the required hormones. A treatment group in which castrated males were given implants of both E2 and DHT would help to resolve this, although this would not get around the problem of dosage or local concentrations. Future experiments should attempt to implant the various hormone concentrations directly into HVC. This has been done for T (Brenowitz and Lent, 2002), but has not been examined for any of its metabolites. The castration of male zebra finches, an oscine species, results in a significant decrease in all courtship behaviors, including but not limited to singing (Harding et al., 1983). In these birds, as well as in castrated male red-winged blackbirds, full reinstatement of sexual behavior requires treatment consisting of both estrogenic and androgenic T metabolites (Harding et al., 1983, 1988). Additionally, blocking ARO in male zebra finches inhibits courtship displays during which a male directs song towards a female (Walters and Harding, 1988). In female canaries, the
inhibition of ARO during T-induced singing decreases song compared with T-treated controls and also decreases the expression of estrogen-sensitive genes such as BDNF in HVC (Fusani et al., 2003). Recently, the pattern of expression of ARO mRNA in the ventromedial telencephalon in wild-caught male song sparrows (Melospiza melodia morphna) was observed to follow the seasonal patterns in aggressive behaviors in this species, indicating that aromatization of T to E2 may be important to the control of aggression as well (Soma et al., 2003). Inhibition of ARO in breeding male song sparrows decreases the volume of HVC, and treatment of these males with estrogen partially rescues this effect (Soma et al., 2003, 2004). In light of these results, and those presented here, it seems likely that activation of singing behavior and seasonal neuroplasticity in songbirds requires the synergistic actions of T and its metabolites to activate the full complement of springlike changes in brain and behavior. It is worth noting that the increase in HVC size observed with T administration was associated with a concomitant increase in singing behavior. The methods described here do not address the question of direction of causality. It is possible that administration of T leads to an increase in HVC and a subsequent increase in singing behavior. It is also possible, however, that T indirectly affects the volume of HVC by altering the bird’s motivation to engage in song, and that it is these types of activity-dependent changes that lead to the increase in HVC size. We attempted to examine this question in this experiment using an ANCOVA to look at the effects of steroid hormone treatment when song rate is considered as a covariate. We did not find any significant effects using this procedure, possibly because of the small number of singers (only the T-implanted males out of all groups) we observed overall. Alternatively, the fact that treatments with steroids no longer had a significant effect on HVC volume when song rate was used as covariate in the ANCOVA would actually be consistent with the second of the interpretations presented above (T indirectly affects HVC size through alterations in singing rates). Recent work into this question of causality has indicated that singing behavior can drive changes in the volumes of song control nuclei independently of the presence of T (for example, see Alvarez-Borda and Nottebohm, 2002; Ball et al., 2002; Sartor and Ball, in press; Sartor et al., 2002). Based on the data presented here, and in light of those described in Tramontin et al. (2000), we conclude that there is in fact a rapid and sequential effect of T on brain and behavior such that activation of song and a motor nucleus involved in song production, HVC, occurs quite rapidly, on the order of 7–10 days, and can occur even in the absence of long, springlike daylengths. On the other hand, the telencephalic song control nuclei RA and Area X, both efferent targets of HVC, require longer exposure to T (and may potentially require exposure to long daylengths) in order for full seasonal recrudescence to be observed. These data suggest that the hormonal effects on reproductive behavior
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do not require large volume increases in all the nuclei known to control that behavior, and challenge the assumption that volumetric changes in the brain are necessary to produce immediate changes in the behavior those areas are known to affect.
Acknowledgments We would like to thank Didier Appeltants and Christel Dejace for assistance with canary maintenance and processing of brain tissue. This work was supported by a grant from the NIH/NINDS (R01 35465). JJS was supported by a postgraduate PGS-B grant from the National Science and Engineering Research Council, Canada.
References Alvarez-Borda, B., Nottebohm, F., 2002. Gonads and singing play separate, additive roles in new neuron recruitment in adult canary brain. J. Neurosci. 22, 8684 – 8690. Appeltants, D., Ball, G.F., Balthazart, J., 2003. Song activation by testosterone is associated with an increased catecholaminergic innervation of the song control system in female canaries. Neuroscience 121, 801 – 814. Arnold, A.P., Nottebohm, F., Pfaff, D.W., 1976. Hormone concentrating cells in vocal control areas of the brain of the zebra finch (Poephila guttata). J. Comp. Neurol. 165, 487 – 512. Ball, G.F., 1999. The neuroendocrine basis of seasonal changes in vocal behavior among songbirds. In: Hauser, M.D., Konishi, M. (Eds.), The Design of Animal Communication. MIT Press, Cambridge, MA, pp. 213 – 253. Ball, G.F., Balthazart, J., 2002. Neuroendocrine mechanisms regulating reproductive cycles and reproductive behavior in birds. In: Pfaff, D.W., et al., (Eds.), Hormones, Brain and Behavior, vol. 2. Academic Press, San Diego, CA, pp. 649 – 798. Ball, G.F., Riters, L.V., Ball, G.F., 2002. Neuroendocrinology of song behavior and avian brain plasticity: multiple sites of action of sex steroid hormones. Front. Neuroendocrinol. 23, 137 – 178. Ball, G.F., Auger, C.J., Bernard, D.J., Charlier, T.D., Sartor, J.J., Riters, L.V., Balthazart, J., 2004. Seasonal plasticity in the song control system: multiple sites of steroid hormone action and the importance of variation in song behavior. In: Zeigler, H.P., Marler, P. (Eds.), Behavioral Neurobiology of Birdsong, Ann. N.Y. Acad. Sci. vol. 1016. The New York Academy of Sciences, New York, pp. 586 – 610. Balthazart, J., Hirschberg, D., 1979. Differential release of various androgens from silastic implants. IRCS Med. Sci. 7, 123. Balthazart, J., Schumacher, M., Malacarne, G., 1985. Interaction of androgens and estrogens in the control of sexual behavior in male Japanese quail. Physiol. Behav. 35, 157 – 166. Balthazart, J., Foidart, A., Wilson, E.A., Ball, G.F., 1992. Immunocytochemical localization of androgen receptors in the male songbird and quail brain. J. Comp. Neurol. 317, 407 – 420. Balthazart, J., Absil, P., Foidart, A., Houbart, M., Harada, N., Ball, G.F., 1996. Distribution of aromatase-immunoreactive cells in the forebrain of zebra finches (Taeniopygia guttata): implications for the neural action of steroids and nuclear definition in the avian hypothalamus. J. Neurobiol. 31, 129 – 148. Bernard, D.J., Wilson, F.E., Ball, G.F., 1997. Testis-dependent and -independent effects of photoperiod on volumes of song control nuclei in American tree sparrows (Spizella arborea). Brain Res. 760, 163 – 169.
475
Bernard, D.J., Bentley, G.E., Balthazart, J., Turek, F.W., Ball, G.F., 1999. Androgen receptor, estrogen receptor a, and estrogen receptor h show distinct patterns of expression in forebrain song control nuclei of European starlings. Endocrinology 140, 4633 – 4643. Brenowitz, E.A., Lent, K., 2001. Afferent input is necessary for seasonal growth and maintenance of adult avian song control circuits. J. Neurosci. 21, 2320 – 2329. Brenowitz, E.A., Lent, K., 2002. Act locally and think globally: intracerebral testosterone implants induce seasonal-like growth of adult avian song control circuits. Proc. Natl. Acad. Sci. U. S. A. 99, 12421 – 12426. Brown, S.D., Johnson, F., Bottjer, S.W., 1993. Neurogenesis in adult canary telencephalon is independent of gonadal hormone levels. J. Neurosci. 13, 2024 – 2032. Dawson, A., King, V.M., Bentley, G.E., Ball, G.F., 2001. Photoperiodic control of seasonality in birds. J. Biol. Rhythms 116, 365 – 380. DeVoogd, T., Nottebohm, F., 1981. Gonadal hormones induce dendritic growth in the adult avian brain. Science 214, 202 – 204. Eens, M., 1997. Understanding the complex song of the European starling: an integrated ethological approach. Adv. Study Behav. 26, 355 – 434. Feare, C.J., 1984. The starling. Oxford University Press, Oxford, UK. Fusani, L., Metzdorf, R., Hutchison, J.B., Gahr, M., 2003. Aromatase inhibition affects testosterone-induced masculinization of song and the neural song system in female canaries. J. Neurobiol. 54, 370 – 379. Follett, B.K., 1984. Birds. In: Lamming, G.E. (Ed.), Marshall’s Physiology of Reproduction. Longman Greene, Edinburgh, pp. 283 – 350. Gahr, M., Gqttinger, H.-R., Kroodsma, D.E., 1993. Estrogen receptors in the avian brain: survey reveals general distribution and forebrain areas unique to songbirds. J. Comp. Neurol. 327, 112 – 122. Harding, C.F., 1986. The role of androgen metabolism in the activation of male behavior. In: Komisaruk, B.R., Siegel, H.I., Cheng, M.F., Feder, H.H. (Eds.), Reproduction: A Behavioral and Neuroendocrine Perspective, Ann. N.Y. Acad. Sci. vol. 474. The New York Academy of Sciences, New York, pp. 371 – 378. Harding, C.F., Sheridan, K., Walters, M.J., 1983. Hormonal specificity and activation of sexual behavior in male zebra finches. Horm. Behav. 17, 111 – 133. Harding, C.F., Walters, M.J., Collado, D., Sheridan, K., 1988. Hormonal specificity and activation of social behavior in male red-winged blackbirds. Horm. Behav. 22, 402 – 418. Johnson, F., Bottjer, S.W., 1993. Hormone-induced changes in identified cell populations of the higher vocal center in male canaries. J. Neurobiol. 24, 400 – 418. Kirn, J.R., Clower, R.P., Kroodsma, D.E., DeVoogd, T.J., 1989. Songrelated brain regions in the red-winged blackbird are affected by sex and season but not repertoire size. J. Neurobiol. 20, 139 – 163. Kreutzer, M.L., Vallet, E.M., 1991. Differences in the responses of captive female canaries to variation in conspecific and heterospecific songs. Behaviour 117, 106 – 116. Leboucher, G., Kreutzer, M., Dittami, J., 1994. Copulation–solicitation displays in female canaries (Serinus canaria): are oestradiol implants necessary? Ethology 97, 190 – 197. Leitner, S., Voigt, C., Garcia-Segura, L.M., Van’t Hof, T., Gahr, M., 2001. Seasonal activation and inactivation of song motor memories in wild canaries is not reflected in neuroanatomical changes of forebrain song areas. Horm. Behav. 40, 160 – 168. Murton, R.K., Westwood, N.J., 1977. Avian breeding cycles. Clarendon Press, Oxford. Nicholls, T.J., Goldsmith, A.R., Dawson, A., 1988. Photorefractoriness in birds and comparison with mammals. Physiol. Rev. 68, 133 – 176. Nottebohm, F., 1980. Testosterone triggers growth of brain vocal control nuclei in adult female canaries. Brain Res. 189, 429 – 436. Nottebohm, F., 1981. A brain for all seasons: cyclical anatomical changes in song-control nuclei of the canary brain. Science 214, 1368 – 1370. Nottebohm, F., Nottebohm, M., Crane, L.A., 1986. Developmental and seasonal changes in canary song and their relation to changes in anatomy of song control nuclei. Behav. Neural Biol. 46, 445 – 471.
476
J.J. Sartor et al. / Hormones and Behavior 47 (2005) 467–476
Nottebohm, F., Nottebohm, M.E., Crane, L.A., Wingfield, J.C., 1987. Seasonal changes in gonadal hormone levels of adult male canaries and their relation to song. Behav. Neural Biol. 47, 197 – 211. Reiner, A., Perkel, D.J., Bruce, L.L., Butler, A.B., Csillag, A., Kuenzel, W., Medina, L., Paxinos, G., Shimizu, T., Striedter, G., Wild, M., Ball, G.F., Durand, S., Gqntqrkqn, O., Lee, D.W., Mello, C.V., Powers, A., White, S.A., Hough, G., Kubikova, L., Smulders, T.V., Wada, K., Dugas-Ford, J., Husband, S., Yamamoto, K., Yu, J., Siang, C., Jarvis, E.D., 2004. Revised nomenclature for avian telencephalon and some related brainstem nuclei. J. Comp. Neurol. 473, 377 – 414. Riters, L.V., Eens, M., Pinxten, R., Ball, G.F., 2002. Seasonal changes in the densities of a2-noradrenergic receptors are inversely related to changes in testosterone and the volumes of song control nuclei in male European starlings. J. Comp. Neurol. 444, 63 – 74. Sartor, J.J., Ball, G.F., 2005. Social suppression of song is associated with a reduction in volume of a song control nucleus in European starlings (Sturnus vulgaris). Behav. Neurosci. 119 (in press). Sartor, J.J., Charlier, T., Pytte, C.L., Balthazart, J., Ball, G.F., 2002. Converging evidence that song performance modulates seasonal changes in the avian song control system. Abstr.-Soc. Neurosci. 28, abstract no. 781.10 (CD-ROM: Program No. 781.10). Schlinger, B.A., Brenowitz, E.A., 2002. Neural and hormonal control of birdsong. In: Pfaff, D.W., et al., (Eds.), Hormones, Brain and Behavior, vol. 2. Academic Press, New York, pp. 799 – 839. Schumacher, M., Balthazart, J., 1983. The effects of testosterone and its metabolites on sexual behavior and morphology in male and female Japanese quail. Physiol. Behav. 30, 335 – 339. Shen, P., Schlinger, B.A., Campagnoni, A.T., Arnold, A.P., 1995. An atlas of aromatase mRNA expression in the zebra finch brain. J. Comp. Neurol. 360, 172 – 184. Smith, G.T., Brenowitz, E.A., Prins, G.A., 1996. Use of PG-21 immunocytochemistry to detect androgen receptors in the songbird brain. J. Histochem. Cytochem. 44, 1075 – 1080.
Soma, K.K., Sullivan, K., Wingfield, J.C., 1999. Combined aromatase inhibitor and antiandrogen treatment decreases territorial aggression in a wild songbird during the nonbreeding season. Gen. Comp. Endocrinol. 115, 442 – 453. Soma, K.K., Schlinger, B.A., Wingfield, J.C., Saldanha, C.J., 2003. Brain aromatase, 5 alpha-reductase, and 5 beta-reductase change seasonally in wild male song sparrows: relationship to aggressive and sexual behavior. J. Neurobiol. 56, 209 – 221. Soma, K.K., Tramontin, A.D., Featherstone, J., Brenowitz, E.A., 2004. Estrogen contributes to seasonal plasticity of the adult avian song control system. J. Neurobiol. 58, 413 – 422. Tramontin, A.D., Hartman, V.N., Brenowitz, EA., 2000. Breeding conditions induce rapid and sequential growth in adult avian song control circuits: a model of seasonal plasticity in the brain. J. Neurosci. 20, 854 – 861. Tramontin, A.D., Perfito, N., Wingfield, J.C., Brenowitz, E.A., 2001. Seasonal growth of song control nuclei precedes seasonal reproductive development in wild adult song sparrows. Gen. Comp. Endocrinol. 122, 1 – 9. Tramontin, A.D., Wingfield, J.C., Brenowitz, E.A., 2003. Androgens and estrogens induce seasonal-like growth of song nuclei in the adult songbird brain. J. Neurobiol. 57, 130 – 140. Walters, M.J., Harding, C.F., 1988. The effects of an aromatization inhibitor on the reproductive behavior of male zebra finches. Horm. Behav. 22, 207 – 218. Wilson, F.E., Donham, R.S., 1988. Daylength and control of seasonal reproduction in male birds. In: Stetson, M.H. (Ed.), Processing of Environmental Information in Vertebrates. Springer-Verlag, Berlin, pp. 101 – 120. Wingfield, J.C., Kenagy, G.J., 1991. Natural regulation of reproductive cycles. In: Pang, P., Schreibman, M. Vertebrate Endocrinology: Fundamentals and Biomedical Implications, vol. 1. Academic Press, New York, pp. 181 – 241.
Coordinated and dissociated effects of testosterone on singing behavior and song control nuclei in canaries (Serinus canaria) Jennifer J. Sartora,*, Jacques Balthazartb, Gregory F. Balla a
Department of Psychological and Brain Sciences, Ames Hall, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA b Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology, University of Lie`ge, 17 Place Delcour (Bat. L1), B-4020 Lie`ge, Belgium Received 28 September 2004; revised 1 December 2004; accepted 3 December 2004
Abstract Temperate zone songbirds that breed seasonally exhibit pronounced differences in reproductive behaviors including song inside and outside the breeding season. Springlike long daylengths are associated with increases in plasma testosterone (T) concentrations, as well as with increases in singing and in the volume of several brain nuclei known to control this behavior. The mechanisms whereby T can induce changes in behavior and brain, and whether or not these effects are differentially regulated, have recently begun to be examined, as has the question of the relative contributions of T and its androgenic and estrogenic metabolites to the regulation of this seasonal behavioral and neural plasticity. In this experiment, we examined the effects of T, 5a-dihydrotestosterone, or 17h-estradiol treatment on castrated male canaries housed on short days and compared neural and behavioral effects in these males to similarly-housed males given only blank implants. We observed that only T treatment was effective in eliciting significant increases in singing behavior after 11 days of hormone exposure. In addition, T alone was effective in increasing the volume of a key song production nucleus, HVC. However, at this time, none of the steroids had any effects on the volumes of two other song control nuclei, Area X of the medial striatum and the robust nucleus of the arcopallium (RA), that are efferent targets of HVC, known to be regulated by androgen in canaries and also to play a role in the control of adult song. T can thus enhance singing well before concomitant androgen-induced changes in the song control system are complete. D 2004 Elsevier Inc. All rights reserved. Keywords: Songbird; Birdsong; Neural plasticity; Seasonal; Estrogen; DHT; Area X; RA; Activity-dependent plasticity
Introduction In temperate zone birds, reproductive behaviors follow an annual cycle consisting of periods of breeding and nonbreeding. Courtship and reproductive behaviors thus occur in most species in the spring, when temperatures are relatively mild and food availability is highest (reviewed in Wingfield and Kenagy, 1991). Changes in photoperiod provide a valuable cue from the environment that allows animals to assess the time of year. The ability to detect and respond to photoperiodic changes has evolved in many avian species living in either the tropics or the temperate
* Corresponding author. Fax: +1 410 516 4478. E-mail address: [email protected] (J.J. Sartor). 0018-506X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2004.12.004
zone (Dawson et al., 2001; Murton and Westwood, 1977; Nicholls et al., 1988; Wilson and Donham, 1988). Physiological responses to photoperiod are mediated by a biological system that includes both a neural as well as an endocrine component (Follett, 1984), which is characterized by seasonal changes in the size of the gonads as well as the blood plasma levels of sex steroid hormones such as testosterone (T; reviewed in Ball and Balthazart, 2002). In addition, springtime increases in testis volume and plasma T concentration are associated with increases in courtship behaviors, of which song is a major component in males of many species of temperate-zone, seasonally-breeding songbirds (see Ball et al., 2004 for review). Male canaries (Serinus canaria), like many temperate zone oscines, exhibit seasonal changes in the volumes of many song control nuclei involved in song production such
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as HVC and the robust nucleus of the arcopallium (RA; for a discussion of the new avian telencephalic nomenclature see Reiner et al., 2004) as well as in Area X of the medial striatum, which is involved in song learning and the contextual production of song (Nottebohm, 1981; reviewed in Ball, 1999; Schlinger and Brenowitz, 2002; although see Leitner et al., 2001). Testosterone is a major mediator of these variations. Laboratory studies of canaries have demonstrated that volumes of the song control nuclei are large when T concentrations in the blood are high, and these nuclei are smaller when T concentrations are decreased (Nottebohm et al., 1986, 1987). Testosterone can be metabolized into the non-aromatizable androgen 5a-dihydrotestosterone (DHT) by the enzyme 5a-reductase, or into estradiol (E2) by the enzyme aromatase (ARO; see Ball and Balthazart, 2002; Harding, 1986 for reviews). Both androgen (Arnold et al., 1976; Balthazart et al., 1992; Bernard et al., 1999; Smith et al., 1996; Soma et al., 1999) and estrogen (Bernard et al., 1999; Gahr et al., 1993) receptors are located within specific song control nuclei such as HVC in canaries and other species. Furthermore, HVC is also surrounded by high expression and activity of ARO (Balthazart et al., 1996; Shen et al., 1995), implying that not only androgens but also estrogens could have important actions in this nucleus. The song production nucleus RA is a target for androgens but contains no receptors for estrogenic metabolites (Balthazart et al., 1992; Bernard et al., 1999). Given this distribution of steroid hormone receptors and metabolizing enzymes in the songbird telencephalon and song control nuclei, it is possible that the mediation of seasonal volumetric changes by T could be dependent on one of its metabolites, or could result from a synergistic action of T and its metabolites at the various sites. In ovariectomized female canaries treated with either T, DHT, or E2, singing was only observed in T-treated females, and dendritic branching in RA was longer in T-treated females than in females from any other hormone treatment group (DeVoogd and Nottebohm, 1981), implying that androgenic and estrogenic metabolites on their own cannot induce these changes, but that these changes may require synergistic effects. In other species of songbirds, it has in fact been shown that both singing as well as changes in morphology of song control nuclei is dependent on the synergistic actions of androgens and estrogens. Castrated male zebra finches (Taeniopygia guttata; Harding et al., 1983; Walters and Harding, 1988) and red-winged blackbirds (Agelaius pheoniceus; Harding et al., 1988) do not engage in sexual behaviors and restoration of these behaviors requires treatment with both androgens and estrogens to restore the full complement including song. In Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii), however, treatment of castrated males with either androgens or estrogens, or a combination of both, was sufficient to elevate the volumes of HVC, RA, and Area X above those for controls without significantly elevating singing behavior (Tramontin et al., 2003).
The sequence and time course of T action on brain and singing behavior are still poorly understood. Recently, however, studies in free-living songbird species have indicated that there could be a dissociation between the behavioral and neural actions of T, such that T may not act uniformly throughout the song system to activate growth and singing behavior, but may instead activate some aspects more quickly than others. For example, in male wild-caught Gambel’s white-crowned sparrows, exposure to T and long daylengths was sufficient to activate growth of HVC and to instigate changes in singing behavior by 7 days posttreatment (Tramontin et al., 2000). Area X and RA, however, were not significantly larger than in control males until 20 days after initial exposure, and song stereotypy (important for singing in a reproductive context) was not fully developed until 20 days after T and long-day exposure. These results suggest that the song system, and singing behavior, are both stimulated by T but that this occurs in a sequential manner, with HVC and song output changing on a quicker timeline than Area X and RA (Tramontin et al., 2000). Furthermore, activation of HVC by T is sufficient to stimulate the growth of its efferents RA and Area X sequentially, whether T is administered systemically (Brenowitz and Lent, 2001) or implanted directly into HVC in castrates (Brenowitz and Lent, 2002). One implication of these experiments is that complete recrudescence of song control nuclei is not necessary for increases in song production, indicating a dissociation between the effects of T on behavior and the brain nuclei known to control that behavior. Additional lines of evidence have developed in the past several years that indicate a dissociation between the effects of T on song control nuclei and singing behavior. For instance, European starlings (Sturnus vulgaris) sing outside the breeding season, when plasma T concentrations are low (Eens, 1997; Feare, 1984) and volumes of the song control nuclei are smallest (Riters et al., 2002). In this species, then, song production per se is not tightly correlated to complete development of song control nuclei and can therefore occur without recrudescence of these regions. The volumetric development of song control nuclei may in these cases relate more to changes in song stereotypy or complexity. In this study, we examined the onset of the effects of T or its metabolites on singing and on song control nuclei in male canaries that were castrated, implanted with either T, DHT, or E2, and maintained on short daylengths for 11 days. We decided upon this length of time for exposure to the steroids by using the hormonal induction of the behavior we were interested in, singing, to guide our investigation of the hormonal initiation of growth of the nuclei involved in the production of that behavior. T-treated males began singing 4 days after hormone implantation, and we observed singing behavior for 1 week after that, at which time there was a significantly higher rate of singing in T-treated males compared to any other groups. By housing these males on short days, we were able to examine the effects of steroid
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treatments in the absence of photoperiodic stimulation to discriminate effects due to the steroids from those of the photoperiod per se. Using this procedure, we observed the development of aggressive and song behavior in these canaries for 11 days following steroid hormone implantation and then sacrificed all subjects for the measure of the song control nuclei volumes when song rates had significantly increased in T birds. This experiment demonstrated that T, but not any of its metabolites on their own, acts first to stimulate song behavior and HVC volume but requires longer exposure to induce growth in Area X and RA, even in the absence of stimulating long daylengths. High rates of singing behavior precede RA and Area X growth, indicating that hormonal effects on behavior do not require increased volumes of all song control nuclei, and demonstrating that changes in a behavior are not necessarily preceded by changes in the volumes of several of the nuclei known to be involved in the control of that behavior.
Materials and methods Experimental animals Twenty adult male canaries were obtained from a breeder in Lie`ge, Belgium and housed indoors in plastic cages (49 95 51 cm) in groups of 4 in the laboratory of Dr. Jacques Balthazart at the Universite´ de Lie`ge on an 8 L:16 D (light/ dark) light cycle. After 5 weeks, all males were castrated under general anesthesia induced by injection of 0.1 ml of a mixture containing 4.33 ml saline solution, 0.33 ml xylazine (Rompun; 20 mg/ml), and 2.10 ml ketamine (Ketalar; 50 mg/ ml). All testes were regressed in these males at the time of castration, indicating that males were in a nonbreeding condition. The two testes were removed through a unilateral incision made posterior to the last rib along the left flank. Forceps were placed at the base of each testis and the whole testis was removed from each side in one motion. The incision was then sutured closed and males were allowed to recover before being returned to their home cages. Following recovery, males were housed an additional 8 weeks on 8 L:16 D in groups of four to ensure that they would respond to the hormone treatments when they were administered. After 8 weeks, all males were implanted subcutaneously with one Silastic implant (Dow Corning, Midland, MI, USA, no. 602-175; 0.76 mm inner diameter, 1.65 mm outer diameter) filled to a length of 10 mm with either crystalline T, DHT, or E2, or left empty as control (blank implants). The doses of hormone used (length and diameter of implants) were selected based on previously published literature indicating that T implants of this size are able to activate singing and to increase the sizes of song control nuclei in canaries to levels characteristic of normal, gonadally-intact males (Appeltants et al., 2003; Nottebohm, 1980). We used similar lengths of implants for the androgenic and estrogenic metabolites of T as we did for
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T itself. It should be remembered however that the release of steroids from Silastic tubing varies inversely with polarity, such that more polar steroids such as E2 diffuse much more slowly, resulting in lower doses from the same lengths of tubing (Balthazart and Hirschberg, 1979). In addition, given that central actions of E2 and to some extent DHT as well are largely controlled by the local metabolism of T at the site of action (reviewed in Ball and Balthazart, 2002), the relevant physiological concentration for estrogenic and androgenic metabolites of T that must be considered is not in the plasma but in the brain. There are no data available indicating the local brain concentrations that will be produced by different implant sizes. It is clear however that the sizes selected here for E2 and DHT implants are able and actually required to activate aspects of reproductive behaviors in a variety of avian species (Balthazart et al., 1985; Kreutzer and Vallet, 1991; Leboucher et al., 1994; Schumacher and Balthazart, 1983). In canaries, the length of E2 implants used has been shown to be physiologically relevant and to elevate E2 to concentrations similar to those seen in photostimulated female canaries (Leboucher et al., 1994). One bird from each cage received one of the four types of implants. Thus, there were 5 birds in each treatment group, with one bird from each group living in each of the five cages for behavioral testing. Males were group-housed in this manner so that aggressive and song behavioral testing could be carried out at the same time for all birds. Two males, one blank-implanted and one T-implanted, died during the behavioral observation period. Behavioral testing All males remained on an 8 L:16 D photoperiod for the duration of behavioral testing, so that the effects of steroid hormone treatment could be examined in the absence of any other cues characteristic of breeding conditions, particularly long daylengths. Testing commenced at the time the lights turned on in the animal room each day, beginning on the day following implantation, and continued for 2 h each day for 10 days. In one observation hour, singing behavior was scored. Each cage was observed for a total of 10 continuous minutes and singing was scored every time it occurred for all males. This method of behavioral observation has been previously shown to be effective for capturing rates of these behaviors in canaries (see Appeltants et al., 2003). In the second hour of observation, singing was again recorded in the same fashion. In addition, during this hour, aggressive behaviors, including chasing, pecking, fighting, and the adoption of aggressive postures (in which males would crouch with wings waving and bills open towards other males), were scored throughout the observation period for all birds. Behavioral observations were concluded and brains were collected when high rates of singing had been observed in some males for a period of 7 days. Singing began on Day 4 of testing and males were therefore sacrificed 11 days following hormone treatment. The order
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of observation sessions was counterbalanced across days of testing.
Results Behavioral characterizations
Perfusions All 20 males from the experiment were perfused on the same day, immediately following behavioral data collection. Once behavioral testing was completed, males were sacrificed one cage at a time, in a random order within the cage, and brains were removed and stored for volume reconstruction of the song control nuclei. Presence of an implant was verified in each bird at the time of sacrifice. Prior to perfusion, males were injected with 0.03 ml of heparin (20 mg/ml, i.m.) and deeply anesthetized with 0.2 ml of the anesthetic mixture described above. An additional 0.03 ml of heparin was injected directly into the heart and males were perfused through the left ventricle with approximately 80 ml of 0.9% saline, followed by 200 ml of fixative (4% paraformaldehyde (PAF) in 0.1 M phosphate buffer, pH 7.2). Brains were dissected out of the skull and postfixed overnight in PAF. The brains were transferred into a sucrose solution (30% sucrose in 4% PAF) for an additional two nights. Following this, brains were frozen on dry ice and stored at 708C until processed for volume reconstruction. Frozen brains were cut in five series in coronal sections at 30 Am using a cryostat. All sections were collected into 1ml Eppendorf tubes containing 4% PAF and stored at 48C until processed. The first series was collected for Nissl staining; the remaining four series of sections were set aside and saved for procedures not described here.
To investigate possible differences in singing behavior for males in the different steroid hormone treatments, the data were analyzed using a one-way analysis of variance (ANOVA) comparing song rates in the four groups of males. The results of this analysis revealed a significant difference among the four conditions (F(3, 14) = 23.46; P b 0.0001; Fig. 1A). Fisher’s protected LSD tests revealed that song rates were significantly higher in T-treated males compared to all other groups (P b 0.001 for each comparison). No other differences between groups were detected. Singing behavior began 4 days after hormone treatment, and Fig. 1B shows the increase in singing
Song nuclei volume reconstruction Brain tissue was Nissl-stained using Thionin. The volume of the song control nuclei HVC, RA, and Area X were calculated for each bird using a Zeiss Axioscope microscope with a CCD camera connected to a Macintosh computer. Each of these nuclei is denser in Nissl-stained material than the surrounding tissue and the boundaries can be easily identified. Toward the caudal end of HVC, this nucleus has been subdivided into an binclusiveQ part and an bexclusiveQ part located medially along the lateral ventricle (Kirn et al., 1989). For the purposes of volume reconstruction in this report, we measured only the binclusiveQ HVC. This also means that we did not include para-HVC sensu Johnson and Bottjer (1993). The brain images were digitized and the area of each nucleus was traced on the digitized section using the program NIH Image (Version 1.62, Wayne Rasband, Bethesda, MD; for HVC only), or the program OpenLab (version 2.0, Improvision Inc.). Area measurements were made bilaterally through the rostral to caudal extent of the nucleus by tracing its perimeter. To derive volume estimates, areas were summed and multiplied by the sampling interval, in this case 0.150 mm. No systematic asymmetries were found, and all reported values represent the mean of the two sides for each nucleus.
Fig. 1. Effects of different steroid hormone treatments on singing behaviors in castrated canaries housed on short (8 L:16 D) photoperiods. Error bars represent standard errors in both cases. (A) Only treatment with T produced singing behavior, and this increase with T exposure was significant compared to all other groups. The asterisk indicates a significant difference in singing behavior at P b 0.05. (B) Song rate increased with T treatment within 4 days following implantation, and remained high for the duration of the experiment. No metabolites of T were effective at elevating song rate above castrated values. Bl = blank implant.
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behavior across the test days, which was only observed in the groups treated with T and DHT. All aggressive behaviors were summed for each male for each testing session and analyzed as one measure. Testosterone-treated males were the one group who exhibited any appreciable amount of behavior, but this difference was not statistically significant due to large standard errors in each group (F(3, 14) = 0.93; P N 0.4; Fig. 2). In the males exhibiting aggression, behaviors largely included chasing of other males, with some pecking and fighting, and very little aggressive posturing. Steroid hormone treatment and HVC volume One-way ANOVA revealed a significant difference in HVC volumes among the four treatment groups (F(3, 14) = 4.38; P b 0.03). Fisher’s protected LSD tests indicated that the volume of the song production nucleus HVC was significantly larger in T-implanted male canaries than in all other groups (P b 0.02 for all comparisons; Fig. 3A). No other groups differed from any other. We also used an analysis of covariance (ANCOVA) with HVC volume as the dependent measure, steroid hormone treatment as the categorical variable, and song rate as the continuous predictor to look for effects of the hormone treatments when song rate is presumed to covary. We did not find any significant effects of either hormone implant or song rate on HVC volume using this method. A simple linear regression of song rate (songs/h) onto HVC volume (mm3) revealed that these two variables were significantly correlated (r = 0.61; P b 0.01; Fig. 3B). Steroid hormone treatment and RA volume One-way ANOVA revealed that the volume of the song production nucleus RA was not different among any of the
Fig. 3. Effects of different steroid hormone treatments on the volume of the song production nucleus HVC in castrated male canaries housed on short photoperiods. (A) Only treatment with T was effective in significantly elevating HVC volume above baseline under these conditions. The standard errors of each mean are represented by the error bars, and the asterisk indicates significance at P b 0.05. (B) Song rate (in songs/h) was positively correlated with HVC volume in T-treated male canaries ( P b 0.05).
hormonal treatments (F(3, 14) = 1.03; P N 0.4; Fig. 4A). There was no significant correlation between song rate (songs/hr) and RA volume (mm3) as demonstrated using simple linear regression (r = 0.23; P N 0.3; Fig. 4B). Steroid hormone treatment and Area X volume The volume of nucleus Area X was not different for any of the hormonal conditions when tested using one-way ANOVA (F(3, 14) = 2.77; P N 0.08; Fig. 5A). A simple linear regression of song rate (songs/h) onto Area X volume (mm3) revealed that these two variables were significantly but negatively correlated (r = 0.54; P b 0.03; Fig. 5B). Fig. 2. Effects of different steroid hormone treatments on a variety of aggressive behaviors (see text) in castrated canaries housed on short photoperiods. Although there were no statistically significant differences between the groups, males treated with T tended to show higher rates of aggressive behaviors compared to other treatment groups.
Discussion In seasonally-breeding songbirds, the association between long, springtime daylengths, high levels of blood
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Fig. 4. Effects of different steroid hormone treatments on the volume of the telencephalic song production nucleus RA in castrated male canaries housed on short photoperiods. (A) There was no effect of any of the steroid hormones on the volume of RA. Error bars represent the standard errors of the means. (B) Song rate (in songs/h) was not significantly correlated with RA volume in male canaries.
plasma testosterone, and increases in song rate and the volume of telencephalic nuclei known to control this behavior has long been apparent (see Ball et al., 2004 for review). Specific models for the regulation of seasonal neuroplasticity by T and the mechanism whereby these variables are related have only begun to be investigated. Recent work in wild-caught white-crowned sparrows has indicated that the timeline for the activation of singing behavior by testosterone may differ from that required for T to have effects on the growth of many of the song control nuclei involved in the production of that behavior, which is known to be regulated by T (Tramontin et al., 2000). In castrated males of this species, treatment of males with T and exposure to long days resulted in increases in both HVC size as well as singing behavior within 7 days of treatment administration, whereas increases in the sizes of the efferent targets of HVC, RA, and Area X were not observed until 20
days post-treatment (Tramontin et al., 2000). In the present experiment, we examined volumes of these song control nuclei in castrated male canaries, housed on short days and treated with either T or one of its metabolites, at a time when a full induction of song rate was observed in T-treated subjects. We therefore selected the length of time that these males would be exposed to their hormonal treatments based on the exhibition of the behavior in which we were interested, and then examined the brain nuclei involved in that behavior once singing had been induced. By housing these males on short days throughout the duration of the experiment, we were able to examine the effects of exposure to T and its metabolites on brain and behavior when the only indicator of a bbreeding conditionQ is plasma hormone concentration. Increasing photoperiods may have stimulatory effects on song system growth even in the presence of small gonads and/or low testosterone
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Fig. 5. Effects of different steroid hormone treatments on the volume of the anterior forebrain nucleus Area X in castrated male canaries housed on short photoperiods. (A) There was no effect of any of the steroid hormones on the volume of Area X. Error bars represent the standard errors of the means. (B) Song rate (in songs/h) was negatively correlated with Area X volume in male canaries ( P b 0.05).
concentrations (Bernard et al., 1997; Tramontin et al., 2001). We found further evidence for the model proposed by Tramontin et al. (2000) in white-crowned sparrows and observed that in canaries, short-term T treatment, even in the absence of stimulating daylengths, is sufficient to activate singing behavior and to increase the volume of HVC, but does not have any effect on its targets, RA and Area X. These data demonstrate that T can relatively rapidly activate some aspects of seasonal neural and behavioral plasticity, whereas other aspects require longer exposure times to high titers of this steroid. The lack of effect of any other steroid hormone on volumes of any song control nuclei is in agreement with previous work in hormonetreated canaries, where only T treatment was found to induce singing behavior and the growth of dendritic spines in RA (DeVoogd and Nottebohm, 1981). The effects of T on RA were observed after 4 weeks of T treatment in this case.
Song rate was correlated with HVC size in these birds but was not significantly correlated with RA volume. The correlation between HVC volume and rate of song production agrees with previous work on singing behavior in canaries (Nottebohm et al., 1987). The lack of correlation between singing and RA size is not surprising given that none of the steroid treatments had any effect on RA size. Surprisingly, the volume of Area X was negatively correlated with song rate in these birds. The causal links potentially explaining this negative correlative remain however unclear at present. At the doses used here and under these conditions, treatment of male canaries with only DHT or E2 alone was not sufficient to elicit any significant change in either brain or behavior in this species. It must be noted however that in this experiment we housed the males in groups of 4 with one male from each treatment group in each cage. We chose this experimental design to avoid issues of pseudo-replication
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that would have occurred had we housed birds from the same hormonal condition together. By using this design, we might have established a situation where T-treated males, because they were active singers and more aggressive, may have suppressed these behaviors in the DHT- and E2-treated males with which they were housed. This suppression of behavior could have led to a subsequent lack of growth in song control nuclei in these males and may explain the lack of effect of the T metabolites on song control nuclei growth. It cannot explain, however, the observation that T acts more quickly on behavior and on HVC volume than on other telencephalic song control nuclei. It should be noted however that these housing conditions may have affected the expressions of aggressive behaviors in groups that did not receive T treatment. Another possible explanation for the lack of effect of any hormones other than T could concern the doses used in this experiment. This seems unlikely, however, given that we chose these doses based on previous literature where similar or shorter lengths of implants were shown to elevate blood plasma concentrations of steroid to physiological levels (for example, see Brown et al., 1993; Nottebohm, 1980). It is thus more likely that neither DHT nor E2 treatment alone significantly changed brain and behavior either because of the limited length of time during which animals were exposed to the hormones, or because synergistic actions between estrogenic and androgenic metabolites are required (see below). In addition, it is possible that the process of song activation and/or seasonal increases in HVC volume requires the metabolization of T into either E2, DHT, or both, but that this must occur at the site of action of these hormones to attain an effective concentration of the hormone locally. This would potentially have occurred with the T treatment described here, and could indicate that a synergy between androgenic and estrogenic metabolites of T is necessary to produce changes in both brain and behavior. This would be one way of achieving high local concentrations of the required hormones. A treatment group in which castrated males were given implants of both E2 and DHT would help to resolve this, although this would not get around the problem of dosage or local concentrations. Future experiments should attempt to implant the various hormone concentrations directly into HVC. This has been done for T (Brenowitz and Lent, 2002), but has not been examined for any of its metabolites. The castration of male zebra finches, an oscine species, results in a significant decrease in all courtship behaviors, including but not limited to singing (Harding et al., 1983). In these birds, as well as in castrated male red-winged blackbirds, full reinstatement of sexual behavior requires treatment consisting of both estrogenic and androgenic T metabolites (Harding et al., 1983, 1988). Additionally, blocking ARO in male zebra finches inhibits courtship displays during which a male directs song towards a female (Walters and Harding, 1988). In female canaries, the
inhibition of ARO during T-induced singing decreases song compared with T-treated controls and also decreases the expression of estrogen-sensitive genes such as BDNF in HVC (Fusani et al., 2003). Recently, the pattern of expression of ARO mRNA in the ventromedial telencephalon in wild-caught male song sparrows (Melospiza melodia morphna) was observed to follow the seasonal patterns in aggressive behaviors in this species, indicating that aromatization of T to E2 may be important to the control of aggression as well (Soma et al., 2003). Inhibition of ARO in breeding male song sparrows decreases the volume of HVC, and treatment of these males with estrogen partially rescues this effect (Soma et al., 2003, 2004). In light of these results, and those presented here, it seems likely that activation of singing behavior and seasonal neuroplasticity in songbirds requires the synergistic actions of T and its metabolites to activate the full complement of springlike changes in brain and behavior. It is worth noting that the increase in HVC size observed with T administration was associated with a concomitant increase in singing behavior. The methods described here do not address the question of direction of causality. It is possible that administration of T leads to an increase in HVC and a subsequent increase in singing behavior. It is also possible, however, that T indirectly affects the volume of HVC by altering the bird’s motivation to engage in song, and that it is these types of activity-dependent changes that lead to the increase in HVC size. We attempted to examine this question in this experiment using an ANCOVA to look at the effects of steroid hormone treatment when song rate is considered as a covariate. We did not find any significant effects using this procedure, possibly because of the small number of singers (only the T-implanted males out of all groups) we observed overall. Alternatively, the fact that treatments with steroids no longer had a significant effect on HVC volume when song rate was used as covariate in the ANCOVA would actually be consistent with the second of the interpretations presented above (T indirectly affects HVC size through alterations in singing rates). Recent work into this question of causality has indicated that singing behavior can drive changes in the volumes of song control nuclei independently of the presence of T (for example, see Alvarez-Borda and Nottebohm, 2002; Ball et al., 2002; Sartor and Ball, in press; Sartor et al., 2002). Based on the data presented here, and in light of those described in Tramontin et al. (2000), we conclude that there is in fact a rapid and sequential effect of T on brain and behavior such that activation of song and a motor nucleus involved in song production, HVC, occurs quite rapidly, on the order of 7–10 days, and can occur even in the absence of long, springlike daylengths. On the other hand, the telencephalic song control nuclei RA and Area X, both efferent targets of HVC, require longer exposure to T (and may potentially require exposure to long daylengths) in order for full seasonal recrudescence to be observed. These data suggest that the hormonal effects on reproductive behavior
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do not require large volume increases in all the nuclei known to control that behavior, and challenge the assumption that volumetric changes in the brain are necessary to produce immediate changes in the behavior those areas are known to affect.
Acknowledgments We would like to thank Didier Appeltants and Christel Dejace for assistance with canary maintenance and processing of brain tissue. This work was supported by a grant from the NIH/NINDS (R01 35465). JJS was supported by a postgraduate PGS-B grant from the National Science and Engineering Research Council, Canada.
References Alvarez-Borda, B., Nottebohm, F., 2002. Gonads and singing play separate, additive roles in new neuron recruitment in adult canary brain. J. Neurosci. 22, 8684 – 8690. Appeltants, D., Ball, G.F., Balthazart, J., 2003. Song activation by testosterone is associated with an increased catecholaminergic innervation of the song control system in female canaries. Neuroscience 121, 801 – 814. Arnold, A.P., Nottebohm, F., Pfaff, D.W., 1976. Hormone concentrating cells in vocal control areas of the brain of the zebra finch (Poephila guttata). J. Comp. Neurol. 165, 487 – 512. Ball, G.F., 1999. The neuroendocrine basis of seasonal changes in vocal behavior among songbirds. In: Hauser, M.D., Konishi, M. (Eds.), The Design of Animal Communication. MIT Press, Cambridge, MA, pp. 213 – 253. Ball, G.F., Balthazart, J., 2002. Neuroendocrine mechanisms regulating reproductive cycles and reproductive behavior in birds. In: Pfaff, D.W., et al., (Eds.), Hormones, Brain and Behavior, vol. 2. Academic Press, San Diego, CA, pp. 649 – 798. Ball, G.F., Riters, L.V., Ball, G.F., 2002. Neuroendocrinology of song behavior and avian brain plasticity: multiple sites of action of sex steroid hormones. Front. Neuroendocrinol. 23, 137 – 178. Ball, G.F., Auger, C.J., Bernard, D.J., Charlier, T.D., Sartor, J.J., Riters, L.V., Balthazart, J., 2004. Seasonal plasticity in the song control system: multiple sites of steroid hormone action and the importance of variation in song behavior. In: Zeigler, H.P., Marler, P. (Eds.), Behavioral Neurobiology of Birdsong, Ann. N.Y. Acad. Sci. vol. 1016. The New York Academy of Sciences, New York, pp. 586 – 610. Balthazart, J., Hirschberg, D., 1979. Differential release of various androgens from silastic implants. IRCS Med. Sci. 7, 123. Balthazart, J., Schumacher, M., Malacarne, G., 1985. Interaction of androgens and estrogens in the control of sexual behavior in male Japanese quail. Physiol. Behav. 35, 157 – 166. Balthazart, J., Foidart, A., Wilson, E.A., Ball, G.F., 1992. Immunocytochemical localization of androgen receptors in the male songbird and quail brain. J. Comp. Neurol. 317, 407 – 420. Balthazart, J., Absil, P., Foidart, A., Houbart, M., Harada, N., Ball, G.F., 1996. Distribution of aromatase-immunoreactive cells in the forebrain of zebra finches (Taeniopygia guttata): implications for the neural action of steroids and nuclear definition in the avian hypothalamus. J. Neurobiol. 31, 129 – 148. Bernard, D.J., Wilson, F.E., Ball, G.F., 1997. Testis-dependent and -independent effects of photoperiod on volumes of song control nuclei in American tree sparrows (Spizella arborea). Brain Res. 760, 163 – 169.
475
Bernard, D.J., Bentley, G.E., Balthazart, J., Turek, F.W., Ball, G.F., 1999. Androgen receptor, estrogen receptor a, and estrogen receptor h show distinct patterns of expression in forebrain song control nuclei of European starlings. Endocrinology 140, 4633 – 4643. Brenowitz, E.A., Lent, K., 2001. Afferent input is necessary for seasonal growth and maintenance of adult avian song control circuits. J. Neurosci. 21, 2320 – 2329. Brenowitz, E.A., Lent, K., 2002. Act locally and think globally: intracerebral testosterone implants induce seasonal-like growth of adult avian song control circuits. Proc. Natl. Acad. Sci. U. S. A. 99, 12421 – 12426. Brown, S.D., Johnson, F., Bottjer, S.W., 1993. Neurogenesis in adult canary telencephalon is independent of gonadal hormone levels. J. Neurosci. 13, 2024 – 2032. Dawson, A., King, V.M., Bentley, G.E., Ball, G.F., 2001. Photoperiodic control of seasonality in birds. J. Biol. Rhythms 116, 365 – 380. DeVoogd, T., Nottebohm, F., 1981. Gonadal hormones induce dendritic growth in the adult avian brain. Science 214, 202 – 204. Eens, M., 1997. Understanding the complex song of the European starling: an integrated ethological approach. Adv. Study Behav. 26, 355 – 434. Feare, C.J., 1984. The starling. Oxford University Press, Oxford, UK. Fusani, L., Metzdorf, R., Hutchison, J.B., Gahr, M., 2003. Aromatase inhibition affects testosterone-induced masculinization of song and the neural song system in female canaries. J. Neurobiol. 54, 370 – 379. Follett, B.K., 1984. Birds. In: Lamming, G.E. (Ed.), Marshall’s Physiology of Reproduction. Longman Greene, Edinburgh, pp. 283 – 350. Gahr, M., Gqttinger, H.-R., Kroodsma, D.E., 1993. Estrogen receptors in the avian brain: survey reveals general distribution and forebrain areas unique to songbirds. J. Comp. Neurol. 327, 112 – 122. Harding, C.F., 1986. The role of androgen metabolism in the activation of male behavior. In: Komisaruk, B.R., Siegel, H.I., Cheng, M.F., Feder, H.H. (Eds.), Reproduction: A Behavioral and Neuroendocrine Perspective, Ann. N.Y. Acad. Sci. vol. 474. The New York Academy of Sciences, New York, pp. 371 – 378. Harding, C.F., Sheridan, K., Walters, M.J., 1983. Hormonal specificity and activation of sexual behavior in male zebra finches. Horm. Behav. 17, 111 – 133. Harding, C.F., Walters, M.J., Collado, D., Sheridan, K., 1988. Hormonal specificity and activation of social behavior in male red-winged blackbirds. Horm. Behav. 22, 402 – 418. Johnson, F., Bottjer, S.W., 1993. Hormone-induced changes in identified cell populations of the higher vocal center in male canaries. J. Neurobiol. 24, 400 – 418. Kirn, J.R., Clower, R.P., Kroodsma, D.E., DeVoogd, T.J., 1989. Songrelated brain regions in the red-winged blackbird are affected by sex and season but not repertoire size. J. Neurobiol. 20, 139 – 163. Kreutzer, M.L., Vallet, E.M., 1991. Differences in the responses of captive female canaries to variation in conspecific and heterospecific songs. Behaviour 117, 106 – 116. Leboucher, G., Kreutzer, M., Dittami, J., 1994. Copulation–solicitation displays in female canaries (Serinus canaria): are oestradiol implants necessary? Ethology 97, 190 – 197. Leitner, S., Voigt, C., Garcia-Segura, L.M., Van’t Hof, T., Gahr, M., 2001. Seasonal activation and inactivation of song motor memories in wild canaries is not reflected in neuroanatomical changes of forebrain song areas. Horm. Behav. 40, 160 – 168. Murton, R.K., Westwood, N.J., 1977. Avian breeding cycles. Clarendon Press, Oxford. Nicholls, T.J., Goldsmith, A.R., Dawson, A., 1988. Photorefractoriness in birds and comparison with mammals. Physiol. Rev. 68, 133 – 176. Nottebohm, F., 1980. Testosterone triggers growth of brain vocal control nuclei in adult female canaries. Brain Res. 189, 429 – 436. Nottebohm, F., 1981. A brain for all seasons: cyclical anatomical changes in song-control nuclei of the canary brain. Science 214, 1368 – 1370. Nottebohm, F., Nottebohm, M., Crane, L.A., 1986. Developmental and seasonal changes in canary song and their relation to changes in anatomy of song control nuclei. Behav. Neural Biol. 46, 445 – 471.
476
J.J. Sartor et al. / Hormones and Behavior 47 (2005) 467–476
Nottebohm, F., Nottebohm, M.E., Crane, L.A., Wingfield, J.C., 1987. Seasonal changes in gonadal hormone levels of adult male canaries and their relation to song. Behav. Neural Biol. 47, 197 – 211. Reiner, A., Perkel, D.J., Bruce, L.L., Butler, A.B., Csillag, A., Kuenzel, W., Medina, L., Paxinos, G., Shimizu, T., Striedter, G., Wild, M., Ball, G.F., Durand, S., Gqntqrkqn, O., Lee, D.W., Mello, C.V., Powers, A., White, S.A., Hough, G., Kubikova, L., Smulders, T.V., Wada, K., Dugas-Ford, J., Husband, S., Yamamoto, K., Yu, J., Siang, C., Jarvis, E.D., 2004. Revised nomenclature for avian telencephalon and some related brainstem nuclei. J. Comp. Neurol. 473, 377 – 414. Riters, L.V., Eens, M., Pinxten, R., Ball, G.F., 2002. Seasonal changes in the densities of a2-noradrenergic receptors are inversely related to changes in testosterone and the volumes of song control nuclei in male European starlings. J. Comp. Neurol. 444, 63 – 74. Sartor, J.J., Ball, G.F., 2005. Social suppression of song is associated with a reduction in volume of a song control nucleus in European starlings (Sturnus vulgaris). Behav. Neurosci. 119 (in press). Sartor, J.J., Charlier, T., Pytte, C.L., Balthazart, J., Ball, G.F., 2002. Converging evidence that song performance modulates seasonal changes in the avian song control system. Abstr.-Soc. Neurosci. 28, abstract no. 781.10 (CD-ROM: Program No. 781.10). Schlinger, B.A., Brenowitz, E.A., 2002. Neural and hormonal control of birdsong. In: Pfaff, D.W., et al., (Eds.), Hormones, Brain and Behavior, vol. 2. Academic Press, New York, pp. 799 – 839. Schumacher, M., Balthazart, J., 1983. The effects of testosterone and its metabolites on sexual behavior and morphology in male and female Japanese quail. Physiol. Behav. 30, 335 – 339. Shen, P., Schlinger, B.A., Campagnoni, A.T., Arnold, A.P., 1995. An atlas of aromatase mRNA expression in the zebra finch brain. J. Comp. Neurol. 360, 172 – 184. Smith, G.T., Brenowitz, E.A., Prins, G.A., 1996. Use of PG-21 immunocytochemistry to detect androgen receptors in the songbird brain. J. Histochem. Cytochem. 44, 1075 – 1080.
Soma, K.K., Sullivan, K., Wingfield, J.C., 1999. Combined aromatase inhibitor and antiandrogen treatment decreases territorial aggression in a wild songbird during the nonbreeding season. Gen. Comp. Endocrinol. 115, 442 – 453. Soma, K.K., Schlinger, B.A., Wingfield, J.C., Saldanha, C.J., 2003. Brain aromatase, 5 alpha-reductase, and 5 beta-reductase change seasonally in wild male song sparrows: relationship to aggressive and sexual behavior. J. Neurobiol. 56, 209 – 221. Soma, K.K., Tramontin, A.D., Featherstone, J., Brenowitz, E.A., 2004. Estrogen contributes to seasonal plasticity of the adult avian song control system. J. Neurobiol. 58, 413 – 422. Tramontin, A.D., Hartman, V.N., Brenowitz, EA., 2000. Breeding conditions induce rapid and sequential growth in adult avian song control circuits: a model of seasonal plasticity in the brain. J. Neurosci. 20, 854 – 861. Tramontin, A.D., Perfito, N., Wingfield, J.C., Brenowitz, E.A., 2001. Seasonal growth of song control nuclei precedes seasonal reproductive development in wild adult song sparrows. Gen. Comp. Endocrinol. 122, 1 – 9. Tramontin, A.D., Wingfield, J.C., Brenowitz, E.A., 2003. Androgens and estrogens induce seasonal-like growth of song nuclei in the adult songbird brain. J. Neurobiol. 57, 130 – 140. Walters, M.J., Harding, C.F., 1988. The effects of an aromatization inhibitor on the reproductive behavior of male zebra finches. Horm. Behav. 22, 207 – 218. Wilson, F.E., Donham, R.S., 1988. Daylength and control of seasonal reproduction in male birds. In: Stetson, M.H. (Ed.), Processing of Environmental Information in Vertebrates. Springer-Verlag, Berlin, pp. 101 – 120. Wingfield, J.C., Kenagy, G.J., 1991. Natural regulation of reproductive cycles. In: Pang, P., Schreibman, M. Vertebrate Endocrinology: Fundamentals and Biomedical Implications, vol. 1. Academic Press, New York, pp. 181 – 241.