Song activation by testosterone is associated with an increased catecholaminergic innervation of the song control system in female canaries

Neuroscience 121 (2003) 801– 814

SONG ACTIVATION BY TESTOSTERONE IS ASSOCIATED WITH AN INCREASED CATECHOLAMINERGIC INNERVATION OF THE SONG CONTROL SYSTEM IN FEMALE CANARIES D. APPELTANTS,a J. BALTHAZARTa*

G.

F.

BALLb

AND

ogy of the song control system, testosterone also regulates the catecholaminergic innervation of most telencephalic song control nuclei in canaries. The endocrine regulation of singing may thus involve the neuromodulatory action of specialized dopaminergic and/or noradrenergic projections onto several key parts of the song control system. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved.

a 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 b Department of Psychological and Brain Sciences, Johns Hopkins University, Baltimore, MD 21218, USA

Key words: catecholamine, dopamine, female canary, testosterone, songbird, learned vocalization.

Abstract—In canaries, singing and a large number of morphological features of the neural system that mediates the learning, perception and production of song exhibit marked sex differences. Although these differences have been mainly attributed to sex-specific patterns of the action of testosterone and its metabolites, the mechanisms by which sex steroids regulate brain and behavior are far from being completely understood. Given that the density of immunoreactive catecholaminergic fibers that innervate telencephalic song nuclei in canaries is higher in males, which sing, than in females, which usually do not sing, we hypothesized that some of the effects induced by testosterone on song behavior are mediated through the action of the steroid on the catecholaminergic neurons which innervate the song control nuclei. Therefore, we investigated in female canaries the effects of a treatment with exogenous testosterone on song production, on the volume of song control nuclei, and on the catecholaminergic innervation of these nuclei as assessed by immunocytochemical visualization of tyrosine hydroxylase. Testosterone induced male-like singing in all females and increased by about 80% the volume of two telencephalic song control nuclei, the high vocal center (HVC) and the nucleus robustus archistriatalis (RA). Testosterone also significantly increased the fractional area covered by tyrosine hydroxylase-immunoreactive structures (fibers and varicosities) in most telencephalic song control nuclei (HVC, the lateral and medial parts of the magnocellular nucleus of the anterior neostriatum, the nucleus interfacialis, and to a lesser extent RA). By contrast, testosterone did not affect the catecholaminergic innervation of the telencephalic areas adjacent to HVC and RA. Together these data demonstrate that, in parallel to its effects on song behavior and on the morphol-

Passeriformes, or songbirds, belong to one of the most recently evolved avian orders and are characterized by their elaborate abilities to learn and produce complex vocalizations (see Ball and Hulse, 1998 for discussion). In the brain, these behavioral aptitudes are paralleled by a specialized network of interconnected cytoarchitecturally distinct cell groups, the so-called song control system (SCS; see Brenowitz et al., 1997; Margoliash, 1997; Wild, 1997 for discussion). During the last decade, the SCS has emerged as a powerful model to study the neural correlates of complex cognitive abilities that involve learning, processing of auditory stimuli and motor control (Ball and Hulse, 1998; Nottebohm, 1999; Doupe and Kuhl, 1999; Doupe et al., 2000; Troyer and Bottjer, 2001). Testosterone (T) modulates song behavior and many of the nuclei in the SCS contain cells which express androgen receptors (AR), and in some cases, estrogen receptors of the ␣ type (ER␣). Songbird species have therefore been widely employed in behavioral neuroendocrinology research (Arnold et al., 1976; Bernard et al., 1999; see Arnold, 1990; Bottjer and Johnson, 1997 for discussion). The SCS includes at least two major circuits which consist of a series of nuclei in the telencephalon, mesencephalon and brainstem (see Fig. 1). A caudal pathway, also named the motor pathway, controls song production although some parts of this circuit may also be involved in song learning (see Brenowitz et al., 1997; Margoliash, 1997 for discussion). This pathway includes projections from the thalamic nucleus uvaeformis and the neostriatal nucleus interfacialis (NIf) to the high vocal center (HVC; the acronym serves as its proper name). HVC projects to the nucleus robustus archistriatalis (RA) which in turn projects to the intercollicular complex (ICo). Finally, both RA and ICo project to motor neurons of the XIIth cranial nerve that control muscles of the soundproducing organ, the syrinx, as well as to nucleus retroambigualis and the rostral ventral respiratory group of neurons which coordinate respiratory activity with song production (Wild, 1997). A second circuit, the anterior

*Corresponding author. Tel: ⫹32-4-366-5970; fax: ⫹32-4-366-5971. E-mail address: [email protected] (J. Balthazart). Abbreviations: ANOVA, analysis of variance; AR, androgen receptor; AVT, area ventralis of Tsai; CAergic, catecholaminergic; DA, dopamine; DAergic, dopaminergic; DLM, dorsolateral thalamic nucleus; ER, estrogen receptors; GCt, substantia grisea centralis (mesencephalic central gray); HSD, highest significant difference; HVC, high vocal center; ICo, intercollicular complex; lMAN, lateral part of the magnocellular nucleus of the anterior neostriatum; mMAN, medial part of the magnocellular nucleus of the anterior neostriatum; NA, noradrenaline; NAergic, noradrenergic; NIf, nucleus interfacialis; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline containing 0.3% Triton X-100; RA, nucleus robustus archistriatalis; SCS, song control system; SN, substantia nigra; T, testosterone; TH, tyrosine hydroxylase; TH-ir, immunoreactive for tyrosine hydroxylase; X, Area X.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00496-2

801

802

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

Fig. 1. Schematic drawing in the sagittal plane of the SCS of oscines. The two main pathways are illustrated in this figure. A caudal pathway mainly involved in the regulation of song production is designated by the black arrows. An anterior forebrain pathway that is involved in song learning and recognition, sensory feedback and the maintenance of crystallized adult song, is illustrated by the white arrows. Beside these two main pathways, another connection that involves the medial part of the mMAN that projects onto HVC is indicated by the black arrow. Different levels of shadows have been used to label the telencephalon (high), the mesencephalon (medium) and the brainstem (low). Additional explanations are given in the text and in the list of abbreviations.

forebrain pathway, is involved in song learning and recognition, sensory feedback and in the maintenance of crystallized adult song (see Bottjer and Johnson, 1997; Doupe and Solis, 1997; Margoliash, 1997 for discussion). This pathway also includes HVC which projects to Area X (X) of the parolfactory lobe which in turn projects to the medial part of the dorsolateral thalamic nucleus (DLM). DLM projects to the lateral part of the magnocellular nucleus of the anterior neostriatum (lMAN) which projects both to RA and X. A projection from the medial part of the magnocellular nucleus of the anterior neostriatum (mMAN) to HVC has been also described (Nottebohm et al., 1982; Foster et al., 1997). In most songbird species of the temperate zone such as the canary (Serinus canaria), song activity is different between the sexes and also varies across seasons (e.g. Nottebohm et al., 1987). In the brain, these seasonal differences in vocal behavior are paralleled by differences in the volume of the SCS (see Ball, 1999 for review but also see Leitner et al., 2001). For example, in canaries, males sing while females rarely sing spontaneously (Pesch and Gu¨ttinger, 1985). Although, female canaries have the same interconnected network of song nuclei as the male, there are morphological differences in song nuclei volume, neuron number, spacing and soma size of HVC and RA, dendritic field width and projection length in RA neuron and, neuronal soma size in lMAN and mMAN between the sexes (Nottebohm and Arnold, 1976; Nottebohm, 1980; DeVoogd and Nottebohm, 1981a; DeVoogd et al., 1985; Bottjer and Dignan, 1988; Bottjer and Maier, 1991). These behavioral and brain sex differences are mainly attributed to different activational effects of T and its metabolites in adult males and females. Plasma T levels are indeed

higher in adult males than in females and this endocrine sex difference is thought to control many of these behavioral and neuroanatomical/sex differences (see Bottjer and Johnson, 1997 and Schlinger, 1997 for discussion). T administrated to adult female canaries stimulates male-like song production, abolishes some, but not all, of the previously cited sexual differences in morphology and increases both the number of HVC and lMAN synapses onto RA neurons (Canady et al., 1988), the number of neuronal soma-somatic gap junction in HVC (Gahr and GarciaSegura, 1996) as well as the concentration of cells containing hormone receptors in mMAN (Nottebohm, 1980; DeVoogd and Nottebohm, 1981b; Brenowitz and Arnold, 1990; Bottjer and Maier, 1991). Although numerous studies have demonstrated the important effects of sex hormones on the development and expression of song behavior as well as on the morphology of the SCS, most of the mechanisms by which hormones induce these effects are largely unknown (for a discussion see Schlinger, 1997 but also Rasika et al., 1999). The study of the neurotransmitter systems associated to the SCS and their potential sexual dimorphism may help increase our understanding of how sex steroid hormones influence the morphology of the SCS and subsequently the production of song. Surprisingly, even though various neurotransmitters and neuropeptides have been shown to be anatomically associated with the SCS (see Ball, 1994 for discussion), very few salient sex differences in their expression have been reported (e.g. Bottjer et al., 1997; Gulledge and Deviche, 1999). The catecholaminergic (CAergic) system represents an exception to this rule. Chemical neuroanatomy has demonstrated that several song control nuclei receive specialized CAergic projections and contain high densities of noradrenergic (NAergic) and/or dopaminergic (DAergic) receptors (e.g. in zebra finches, Taeniopygia guttata: Sakaguchi and Saito, 1989; Barclay and Harding, 1988, 1990; Bottjer, 1993; Soha et al., 1996; Mello et al., 1998; Riters and Ball, 2002; in European starlings, Sturnus vulgaris: Ball, 1994; Casto and Ball, 1994; Bernard and Ball, 1995). This strong CAergic innervation exhibits significant sex differences. For example, in male canaries and zebra finches, the density of fibers and terminals immunoreactive for tyrosine hydroxylase (TH-ir), the enzyme which constitutes the rate-limiting step in the biosynthesis of catecholamines is higher in most telencephalic song nuclei when compared with their surrounding telencephalic areas. By contrast, in females, the level of staining for tyrosine hydroxylase (TH) is equal in most song nuclei when compared with their adjacent structures (Bottjer, 1993; Appeltants et al., 2001). Although only a few studies have investigated the function of CAergic inputs to the SCS, these neuromodulatory projections have been proposed to regulate different aspects of song perception and production such as song auditory processing (Appeltants et al., 2002b), attentional processes related to song production (Barclay et al., 1992, 1996) and motor access to auditory feedback (Dave et al., 1998). Most of the mechanisms by which T acts on the SCS to modulate song behavior are largely unknown. Steroids

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

may regulate behaviors not only by acting directly on cells in the brain areas that are important for the behavior of interest but also by acting indirectly (i.e. transsynaptically) on cells at distant brain sites that then project to the site of interest and modify its activity (Beyer and Feder, 1987; Balthazart and Ball, 1995; Ball et al., 2002). The CAergic innervation of the SCS represents a good candidate that could mediate some of the effects of sex steroids on singing. Activation of various types of behaviors through the regulation by steroids of the CAergic transmission has been described in hypothalamic systems controlling sexual behavior (Nock and Feder, 1981; Etgen et al., 1990, 1992) and recent studies performed in mammals also suggest similar mechanisms of hormonal modulation of high levels of information processing in telencephalic areas such as the prefrontal cortex (Kritzer and Kohama, 1998; Adler et al., 1999). In addition, catecholamine cell groups that project to the SCS have been shown to express either AR or ER␣ in canaries (Maney et al., 2001). In the present study, we investigated the potential effects of T in the control of the CAergic innervation of the specialized telencephalic structures involved in song learning and production in songbirds. We demonstrate that treatment with T of adult female canaries activates maletypical singing and increases in parallel the volume of the two main telencephalic song control nuclei, HVC and RA, as well as on the density of TH-ir fibers innervating the different telencephalic song nuclei but not of the surrounding neo-and archistriatal structures.

EXPERIMENTAL PROCEDURES Subjects and neuroendocrine procedures This study was performed on adult female canaries (Serinus canaria) bought from a local dealer in Lie`ge, Belgium (SPRL G. Guisset & Fils) in the fall (October). Birds were maintained in indoor aviaries in unisexual groups with a maximum of 10 subjects per cage under a photoperiod of 11 h of light: 13 h of dark per day, with food and water available ad libitum. In this way we maintained the birds in a photosensitive reproductive state indicative of late winter– early spring (Nicholls et al., 1988). They were randomly assigned to one of two groups that either received a T implant (T-treated group, n⫽8) or an empty implant as control (Control group, n⫽7). T implants were constructed from Silastic tubing (Dow Corning, Midland, MI, USA; no 602–175; 0.76 mm inner diameter; 1.65 mm outer diameter) packed with crystalline T to a length of 10 mm and sealed with Silastic glue (Dow Corning). Based on previously published studies utilizing similar procedures, these implants presumably established in the treated females plasma T levels that are in the high physiological range of values normally observed in sexually mature male canaries (Nottebohm, 1980). Blank implants were sealed at both ends but left empty. Implants were inserted subcutaneously in the flank region on the left side. Once implanted, birds were housed in individual cages (17⫻25.5⫻40 cm) until the end of the experiment. Birds were housed, manipulated, and killed in agreement with the Belgian laws on “Protection and Welfare of Animals” and on the “Protection of Experimental Animals” and the International Guiding Principles for Biomedical Research involving Animals published by the Council for International Organizations of Medical Sciences. The protocols were approved by the Ethics Committee for the Use of Animals at the University of Lie`ge. Experiments were designed to minimize the numbers of subjects and their

803

suffering while maintaining a sufficient statistical power for all comparisons.

Song quantification and physiological data Starting 3 weeks after the implantation of the Silastic capsule, the songs of all subjects were counted and tape recorded (Sony TCM-5000EV) for 45 min in the morning during 5 consecutive days. The mass of each subject as well as the size of the external tip of its cloacal protuberance (cloacal protuberance area⫽length⫻width in millimeters) was assessed before the hormonal treatment was started and 3 days after the last session of song recording, just before the terminal perfusion that took place 4 weeks after the implantation of the Silastic capsule. The mass of the ovary and diameter of the largest follicle were also measured at that time.

Tissue processing Three days after the last song recording, birds were injected with 100 ␮l heparin (20 mg/ml i.m.) before being irreversibly anesthetized with 200 ␮l Hypnodil (Janssen Pharmaceutica, Beerse, Belgium; 2.5 mg/ml). They were then perfused through the heart with a saline solution (0.15 M NaCl) followed by 200 ml fixative (paraformaldehyde 4% in 0.15 M phosphate buffer, pH 7.2). Brains were immediately dissected out of the skull, post-fixed for 1 h in the fixative solution and placed overnight in a 20% sucrose solution in 0.1 M phosphate buffer. On the next day, brains were frozen on powdered dry ice and stored at ⫺75 °C until used. Histological processing of experimental and control brains were then conducted in parallel. Brains were cut in a cryostat in the coronal plane at a thickness of 30 ␮m, starting at the caudal end, with the plane of section adjusted to match as much as possible the atlas of the canary brain (Stokes et al., 1974). One pair of sections was collected every 120 ␮m. The first set of sections was Nissl-stained using Toluidine Blue while the second one was stained by immunocytochemistry for TH.

TH immunocytochemistry Sections were treated for 20 min with 0.6% H2O2 in methanol to block endogenous peroxidase activity. After two rinses in phosphate-buffered saline (PBS; 0.01 M; pH 7.2) and one in PBS containing 0.3% Triton X-100 (PBST), sections were placed for 30 min in blocking solution (goat serum) diluted 1:20 in PBST. Sections were then incubated for 48 h at 4 °C with the primary antibody (mouse monoclonal anti-TH antibody; Incstar, Stillwater, MN, USA) diluted in 1:1000 in PBST. Previous work in a variety of avian species has shown that this antibody specifically recognizes TH in the avian brain (e.g. Bailhache and Balthazart, 1993). Thereafter, sections were incubated for 2 h at room temperature in a biotin-conjugated goat anti-mouse antibody (1:200 in PBST; Dakopatts, Glostrup, Denmark). After three rinses in PBST, sections were incubated for 90 min at room temperature in horseradish-peroxidase-conjugated streptavidin (1:400 in PBST; Dakopatts). After three further rinses in PBST, sections were reacted with 3,3⬘-diaminobenzidine tetrahydrochloride (12 mg in 30 ml PBST containing 12 ␮l 30% H2O2) for 7 min. Sections were mounted on microscope slides in a gelatin medium and coverslipped. Pairs of brains from T-treated and control canaries were systematically stained in parallel.

Data analysis All analyses were performed with the experimenter being blind to the experimental condition of the birds. The volumes of the song nuclei HVC and RA were determined by measuring with an imageanalysis software package (NIH Image; Wayne Rasband, NIH, Bethesda, MD, USA) the area covered by each nucleus in the

804

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

Fig. 2. Photomicrographs illustrating the localization of the areas selected to quantify the relative density of TH-immunoreactive structures in HVC and RA and adjacent structures. (A) HVC in a section stained for TH with the rectangular area selected in the song nucleus (b) and in the adjacent neostriatum (c). (B) Higher magnification of the sampled area in HVC. (C) Higher magnification of the sampled area in the neostriatum adjacent to HVC. (D) RA in a section stained for TH with the rectangular area selected in the song nucleus (e) and in the adjacent archistriatum (f). (E) Higher magnification of the sampled area in RA. (F) Higher magnification of the sampled area in the archistriatum adjacent to RA.

Nissl-stained sections (approximately eight for HVC and five for RA) that had been digitized by a charge-coupled device camera (XC-77 CE; Sony, Japan) connected to a microscope and computer and multiplying these areas by the sampling interval between sections (120 ␮m). All brain nuclei were measured in both hemispheres. The density of TH-ir structures present in HVC and RA was quantified by image analysis separately in each hemisphere. In HVC, as in most song nuclei, the pattern of TH-immunolabeling is essentially uniform throughout the rostro-caudal and medio-lateral extension of the nucleus (Soha et al., 1996; Appeltants et al., 2001). Areas measured in Nissl-stained material to determine HVC volume were used to select the adjacent TH-labeled section where HVC displays the largest cross-sectional area. In this section, two rectangular fields as observed with a 40⫻ objective (250⫻300 ␮m) were captured and digitized with a camera (Sony Power HAD 3CCD Color Video Camera) connected to a microscope (Leica DMRB) and a digital still recorder (Sony; DKR-700/ 700P). One field was located in the center of HVC while the second field was located in the neostriatum just ventral and adjacent to the song control nucleus (see Fig. 2A,B,C). In contrast to other telencephalic song control nuclei, RA only contains a small amount of TH-ir fibers and terminals and their distribution is heterogeneous with a higher density of TH-ir structures being present in the medial and ventral parts of the nucleus (Bottjer, 1993; Soha et al., 1996; Appeltants et al., 2001). Like for HVC, the largest cross-sectional area through RA was identified based on adjacent Nissl-stained sections and two fields as observed with a 40⫻ objective were digitized, one located in the ventro-medial part of RA where the density of TH-ir structures is maximal, one located in the ventro-lateral adjacent archistriatum (see Fig. 2D–F). After transforming these images from “color” into “grayscale” with Adobe Photoshop software, the amount of TH-ir structures in these digitized fields was quantified with NIH Image (Wayne Raband, NIH). The images were first made binary and then the manual threshold function was used to delineate areas immunoreactive for TH from the background. The TH-ir area was then calculated by the program and expressed in pixels for the considered song control nucleus (A) and for the control adjacent area (B). This value was then expressed as a percentage of the total surface that had been analyzed (250⫻300 ␮m, i.e. 437,575 pixels) and labeled Fractional Area covered by immunoreactive structures. We additionally calculated for each subject the differ-

ence between the fractional areas covered by TH-ir structures inside and outside of each song control nucleus to provide an objective measure (Differential Fractional Area) of the higher density of positive structures inside the nuclei by comparison with the surrounding tissue. Because the song nuclei NIf, mMAN, lMAN and X do not show clear cytoarchitectural boundaries in Nissl-stained sections of female brains and are usually not visible by TH immunocytochemistry in control subjects, a similar quantitative analysis could not be performed for these song nuclei. The effects of T on the CAergic fibers and terminals in these nuclei was thus assessed qualitatively in these nuclei by comparing under a microscope (Leica DMRB, Wetzlar, Germany) at low and high magnification the density of TH-ir structures in the nucleus and in the surrounding tissue. When appropriate, the identification of the nuclei was also supported by observation by the adjacent Nissl-stained sections. Based on these observations we determined in each subject whether each nucleus could or could not be detected by a higher or lower density of immunoreactive structures compared with the surrounding tissue. With the exception of area X in a few subjects, none of these nuclei could be detected by a lower level of staining in the nucleus when compared with the surrounding tissue. Photomicrographs of representative brain areas were obtained with a camera (Sony Power HAD 3CCD Color Video Camera) connected to a microscope (Leica DMRB) and to a digital still recorder (Sony; DKR-700/700P).

Statistics Data were analyzed by repeated measures analysis of variance (ANOVA) or t-test. When appropriate, ANOVAs were followed by Tukey highest significant difference (HSD) post hoc comparisons adapted for repeated measures. Semi-quantitative data of the relative CAergic innervation of the song nuclei NIf, mMAN and lMAN were analyzed by the non-parametric ␹2 test. Differences were considered significant for Pⱕ0.05.

RESULTS Morphological and physiological effects of T A two-way ANOVA with one independent (treatments: T and Control) and one repeated (periods: before and after T) factor identified a significant effect of the hormone treat-

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

805

Table 1. Quantitative analysis of the effects of testosterone treatment on various morphological measures in female canariesa Control birds Before T

T-treated birds

After T

Before T

13.5⫾0.7

12.5⫾0.7

12.9⫾0.4

19.1⫾1.1

Body weight, g

21.0⫾0.3

21.1⫾0.3

22.3⫾0.7

22.3⫾0.7

Ovary weight, g a

–

Periods

Inter

12.1 P⬍0.01 2.607 NS –

15.6 P⬍0.01 0.019 NS

31.715 P⬍0.0001 0.052 NS

–

–

t-test

After T

Cloacal Protuberance area, mm2

Follicule diameter, mm

Groups

0.71⫾0.12

0.59⫾0.06

0.026⫾0.005

0.022⫾0.002

–

⫺0.934 NS ⫺0.776 NS

The table presents data (means and S.E.M.) recorded before and after the treatment with T in the control and T-treated groups.

ment (F1,13⫽12.147, P⬍0.01), of the period (F1,13⫽ 15.598, P⬍0.01) and of their interaction (F1,13⫽31.715, P⬍0.0001) on the cloacal protuberance area. Post hoc comparisons (Tukey HSD) indicated that the mean area of the cloacal protuberance in birds treated with T was significantly different before and after hormonal treatment (P⬍0.01) while no difference was detected in the control group between the two periods (P⬎0.05). A similar analysis of the effects of the hormonal treatments on body weight detected no overall effect of the hormone, the period, and their interaction (see Table 1 for detail). Inspection of the gonads after killing confirmed their regressed size. This was expected given that birds were collected in the early fall, a period that follows the mating season. Unpaired t-tests indicated no significant difference between groups for both the diameter of the largest follicle and the total ovary mass (see Table 1). Song activity and song nuclei volume The present study confirmed the well-known effects of T on both song activation and on the volume of the song nuclei, HVC and RA (see Table 2). All birds treated with T produced frequent songs while no singing was recorded in the control group, with the exception of one female that produced a few spontaneous songs. This difference in song occurrence frequency between the two groups was statistically significant (t⫽2.185; P⬍0.05). Song structure in these T-treated females was however much simpler than in normal males (see Fig. 3 for examples of sonograms) Although no systematic analysis of these songs was performed, it is clear that females only sang a limited number of syllables (usually two to four) which is largely less than average males who almost always produce at least 10 –20

different syllables. The females’ songs also appeared shorter than in males. These differences have been previously documented (see Nottebohm, 1980) and they were not analyzed here in detail. T also increased the volume of two song nuclei, HVC and RA. A two-way ANOVA with hormone treatment as independent factor and brain side as repeated factor indicated no significant effect of the brain side on these volumes (HVC, F1,13⫽2,05, P⫽0.176; RA, F1,13⫽0.902, P⫽0.359). Mean of the left and right volumes were thus calculated for each subject and compared by unpaired t-test that demonstrated the presence of significant effects of T on the volumes of HVC (t⫽2.401; P⬍0.05) and RA (t⫽4.390; P⬍0.001). The magnitude of these effects of T on the volumes of HVC and RA is similar to what was reported before in a similar experiment (Nottebohm, 1980). The volume of the nuclei in T-treated females remains however substantially lower (about 50%) than in normal sexually mature males indicating that the masculinization of these morphological features was only partial following exposure to T in adulthood. TH staining in telencephalic song control nuclei HVC and RA could easily be delineated on Nissl-stained material (Fig. 4A, C and Fig. 5A, C). In adjacent sections stained for TH, HVC was difficult or impossible to detect on the basis of TH staining in control females although, in at least two out of seven subjects, this nucleus could be defined by a lower density of fibers compared with the surrounding neostriatum (see Fig. 4B). In contrast, in most T-treated females, HVC was clearly visible based on the higher density of TH-ir fibers in the nucleus when compared with the surrounding telencephalon (see Fig. 4D). In

Table 2. Quantitative analysis of the effects of testosterone treatment on song activation (total occurrence frequencies during the five observation periods of 45 min each) and on the volume of the song nuclei HVC and RA (average of left and right volumes)a

Total song number HVC volume, mm2 RA volume, mm2

Control birds

T-treated birds

t-test

P

3.429⫾3.429 0.165⫾0.045 0.089⫾0.015

53.125⫾20.965 0.300⫾0.035 0.161⫾0.008

2.185 2.401 4.390

P⬍0.05 P⬍0.05 P⬍0.05

a Values presented are means and S.E.M. In the control group, one untreated adult female canary produced spontaneous song which explains the mean different from zero and associated with an equal S.E.M. All data were compared by Student’s t-tests and the corresponding two-tailed probabilities are shown in the last column.

806

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

Fig. 3. Representative sonograms of songs recorded in four T-treated female canaries. Sounds were digitized on Macintosh computers and analyzed by the Sound Edit program (Farallon Computing Inc., Emeryville, CA, USA) providing graphical representations of the changes in sound frequency (between 0 and 10 kHz [K]) as a function of time (bar on the bottom right⫽1 s). One can observe the relatively short duration of these songs and the small number of different syllables that are used.

Fig. 4. Photomicrographs illustrating the song control nucleus HVC in adjacent sections stained for Nissl bodies and TH. (A) HVC in a section stained for Nissl bodies in a control female canary. (B) Adjacent section stained for TH in the same control bird. (C) HVC in a section stained for Nissl bodies in a female canary treated with T. (D) Adjacent section stained for TH in the same T-treated bird.

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

807

Fig. 5. Photomicrographs illustrating the song nucleus RA in adjacent sections stained for Nissl bodies and TH. (A) RA in a section stained for Nissl bodies in a control female canary. (B) Adjacent section stained for TH in the same control bird. (C) RA in a section stained for Nissl bodies in a female canary treated with T. (D) Adjacent section stained for TH in the same T-treated bird.

birds of both groups, the level of TH labeling in HVC was homogenous throughout the rostro-caudal and medio-lateral extension of the nucleus. In control females, RA was difficult to delineate in sections stained by immunocytochemistry for TH (see Fig. 5B). In several T-treated subjects, this round nucleus could in contrast be distinguished from the surrounding archistriatum by a slightly higher density of TH-ir fibers which highlighted mostly the ventro-medial part of the nucleus (see Fig. 5D). The analysis of the fractional area covered by TH-ir structures by a four way-ANOVA with the endocrine treatments (Control and T) as independent factor and three repeated factors, namely, the brain areas (neostriatal tissue including HVC and archistriatal tissue including RA), the location within these areas (HVC versus adjacent neostriatum and RA versus adjacent archistriatum) and the brain sides (right and left) indicated no significant effect of the brain side (F1,13⫽1.299, P⫽0.275) and no interaction of this factor with the other factors included in the analysis (all P⬎0.05). The means of data corresponding to the left and right side were thus calculated and this factor was excluded from the analysis. The resulting three-way ANOVA, summarized in Fig. 6A, indicated a nearly significant overall effect of the treatment (F1,13⫽4.633, P⫽0.0507) but fully significant effects of the brain area (neo- versus archistriatum: F1,13⫽31.067, P⬍0.0001) and of the specific location within these areas (In versus Out of the song control nucleus: F1,13⫽5.189, P⫽0.040). Primary interactions between these factors were also significant namely the interaction between treatments and brain areas (F1,13⫽10.192, P⫽0.007) and between treatments and lo-

cation in/out of the nucleus (F1,13⫽5.046, P⫽0.043). The secondary interaction (treatment by brain areas by location in/out of nucleus) was also close to significance (F1,13⫽3.981, P⫽0.067) suggesting highly localized effects of T on the TH innervation. To further explore the origins of the interactions, data relative to each song nucleus were reanalyzed separately by two way ANOVA with the treatments as independent factor and location in or out of the song control nucleus as repeated factor. When applied to HVC and the adjacent neostriatum, the analysis identified fully significant effects of the treatment (F1,13⫽6.400, P⫽0.025), of the location (F1,13⫽5.269, P⫽0.039) and of their interaction (F1,13⫽5.657, P⫽0.033). The hormonal treatment increased the area covered by TH-ir structures specifically in HVC but not in the adjacent neostriatum. Post hoc comparisons confirmed the significance of a difference between groups in the area covered by TH-ir structures in HVC (P⬍0.05) but there was no group difference in the amount of TH staining in the surrounding neostriatum (P⬎0.05). The amount of TH staining in HVC was also significantly larger than in the neostriatum in T-treated birds (P⬍0.01) but not in control birds (P⬎0.05). The same analysis applied to the area covered by TH-ir structures in RA and the adjacent archistriatum detected no significant effect of the treatment (F1,13⫽0.970, P⫽0.343) and location in or out of the nucleus (F1,13⫽1.737, P⫽0.210) and no interaction between these factors (F1,13⫽1.164, P⫽0.300). The analysis of the Differential Fractional Areas by a two-way ANOVA with the hormonal treatments as the independent factor and nuclei as repeated factor confirmed

808

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814 Table 3. Quantitative analysis of the visibility of the telencephalic song nuclei NIf, IMAN and mMAN based on the pattern of immunoreactivity for TH in the song nucleus when compared to the surrounding structuresa Nuclei

NIf mMAN IMAN

Controls

T-treated

Detect.

N.Detect.

Detect.

N.Detect.

2 1 1 0 1 0

5 5 6 6 6 6

6 6 5 5 6 6

2 2 3 3 2 2

␹2

P

3.233 4.667 3.616 5.833 5.529 7.875

0.072 0.031 0.057 0.016 0.019 0.005

a

The table list the numbers of subjects in which each nucleus was detectable (Detect.) or non-detectable (N. Detect.) based on the differential TH staining. Because there was one singing female in the control group which displayed most of the behavioral and morphological characteristics of the singing T-treated female canaries, analyses were also performed after exclusion of this subject. The corresponding results are always presented in italics in second line. They correspond to the comparisons of the TH innervation between singing and nonsinging females.

Fig. 6. Bar graphs illustrating the effect of T on the fractional areas (A) and differential fractional areas (B) covered by fibers and varicosities immunoreactive for TH in the song nuclei HVC and RA as well as in their respective surrounding structures (neostriatum [Neo] and archistriatum [Archi]). The symbols above the bars in A summarize the results of the Tukey HSD tests comparing fractional areas in control and T-treated subjects (*⫽P⬍0.05; NS⫽P⬎0.05).

the existence of an overall effect of T (F1,13⫽5.046, P⫽0.043) but no overall difference between nuclei (F1,13⫽3.123, P⫽0.101). The interaction between the two factors was nearly significant reflecting the somewhat more pronounced effect of T in HVC than in RA (F1,13⫽3.981, P⫽0.067). The qualitative analysis of the relative densities of TH-ir structures identifying four other song control nuclei, namely, NIf, mMAN, lMAN and area X also identified major effects of the T treatment that proved to be statistically

significant by the ␹2 test (see Table 3). The song control nucleus NIf was difficult if not impossible to identify in Nissl-stained sections. It has however been demonstrated that in adult male canaries and zebra finches, this nucleus can be identified in sections stained for TH by the high density of immunoreactive fibers present in a discrete elongated area of the neostriatum just above the dorsal boundary of the paleostriatum augmentatum (Bottjer, 1993; Soha et al., 1996; Appeltants et al., 2001). This pattern of labeling was observed here in only two out of seven control females, including the female that had produced a few songs during the recording sessions. In contrast, NIf could easily be detected by the higher level of immunoreactive fibers in six of eight T-treated birds (see Fig. 7A). This frequency difference between groups is not significant by the non-parametric ␹2 test (␹2⫽3.233, P⫽0.072) but it becomes significant when the singing control female is excluded from the analysis so that the test focuses on the comparison of singing versus non-singing subjects (␹2⫽4.667, P⬍0.05). As previously observed in control female canaries (Bottjer and Maier, 1991), the song control nuclei mMAN and lMAN were difficult to detect in Nissl-stained sections. The treatment with T however increased the cell size in

Fig. 7. Photomicrographs illustrating the song nuclei Nif (A) and mMAN and lMAN (B) in sections stained for TH in a female canary treated with T.

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

these nuclei so that they became easily identifiable. In sections stained for TH, mMAN could not be detected in control birds, except in one control subject that was singing and was also characterized by a higher level of TH staining in NIf. In this bird and in most T-treated females (five birds out of eight), this nucleus could be identified by a higher level of TH-ir structures than the surrounding tissue (see Fig. 7B). This difference is not fully significant (␹2⫽3.616, P⫽0.057) but becomes significant after exclusion of the control female that was observed singing (␹2⫽5.833, P⬍0.05). lMAN was not detected on the basis of TH staining in control females except for the singing female in which this nucleus exhibited a higher level of staining when compared with the surrounding structures like in six of eight T-treated birds (see Fig. 7B). Most of the TH-ir fibers covering lMAN were organized in small clusters completely surrounding immunonegative cell bodies and thereby forming basket-like structures. ␹2 Tests indicated significant differences between the groups in the presence/absence of dense TH-ir fibers when the singing control bird was included (␹2⫽5.529, P⬍0.05) or excluded from the analysis (␹2⫽7.875, P⫽0.005). In both T-treated and control female canaries, area X displayed levels of TH-ir staining similar to the surrounding lobus parolfactorius, a structure homologous to parts of the basal ganglia in mammals that receives a rich DAergic innervation. In two birds treated with exogenous T, the density of TH-ir structures appeared however slightly weaker in area X than in the surrounding tissue.

DISCUSSION In this study, TH immunocytochemistry was used to investigate the potential effects of T in the regulation the CAergic innervation of the telencephalic areas of the SCS in adult female canaries. As previously reported, T stimulated song production and increased the volume of the song control nuclei, HVC and RA (e.g. Nottebohm, 1980). The masculinization of these behavioral and morphological features was however incomplete as attested by the fact that T-treated females sang simpler and shorter songs that normal sexually active males and that the volume of their HVC and RA was still only about one half of the volume observed in males (Nottebohm, 1980). Whether this relates to the limited duration of the T treatment or to permanent organizational effects of steroids early in life remains unclear at present. In addition, qualitative and quantitative analyses identified statistically significant differences between T-treated and control birds in the amount of TH staining present in most telencephalic song nuclei. These results indicate that, in parallel with its numerous effects on the morphology of the SCS and on song behavior, T also modulates an important aspect of the neurochemical specialization associated to this system. Because the CAergic innervation of most song nuclei is low in non-singing female canaries but high in singing males or T-treated females, we hypothesize that these CAergic inputs to the SCS are critical for the control of

809

song behavior, a notion that is also supported by a limited number of pharmacological studies. T differentially affects the levels of TH in song control nuclei of female canaries The present study confirms that, in adult female canaries, which do not usually sing, most telencephalic song nuclei are innervated by similar amounts of TH-ir fibers and terminals as the surrounding telencephalic tissue (Appeltants et al., 2001). T-treatment however significantly increases the density of these TH-ir structures in most telencephalic song nuclei. The reliability of this finding is largely supported by the facts that a highly specific antibody was used for immunocytochemistry and that sections were processed in matched pairs of control and T-treated birds and then quantified by an observer blind to the experimental treatments to avoid any systematic bias in the results. HVC, a nucleus that plays a key role in song production and possibly in sensory-motor integration during song learning seems to receive the densest CAergic innervation following T treatment. In untreated birds, the density of CAergic fibers and terminals in this nucleus is equal to or even lower than in the surrounding neostriatum but its boundaries are clearly outlined by a high density of TH-ir structures in T-treated females. Similarly lMAN, a nucleus essential for song learning in juveniles and vocal motor plasticity in adults (Benton et al., 1998), could be clearly detected in TH-stained sections in most T-treated females but not in non-singing control birds. A large fraction of the TH-ir fibers present in lMAN was also organized in basketlike structures enwrapping immunonegative somata and proximal parts of the dendrites of their postsynaptic targets. This dense CAergic innervation of specific cells in lMAN suggests important and specific functions for dopamine (DA) and/or noradrenaline (NA) in this song nucleus. T treatment also increased the amount of CAergic fibers and terminals in NIf and mMAN although these effects were less salient than in HVC or lMAN. NIf, which does not display clear cytoarchitectonic boundaries and was originally discovered by retrograde tracing, can be identified by fibers that are immunoreactive for vasoactive intestinal peptide and met-enkephalin in both male and female zebra finches treated or not with T (Ball et al., 1995). TH-ir structures represent another neurochemical marker of this nucleus but only in subjects exposed to high circulating levels of T such as sexually mature males (Appeltants et al., 2001) and T-treated females (present study). The pre-motor song control nucleus RA, which innervates the syrinx and receives projections both from the motor pathway, via HVC, and from the anterior forebrain pathway via lMAN, displays a low level of CAergic innervation compared with the other telencephalic song control nuclei in both male canaries and zebra finches (Bottjer, 1993; Soha et al., 1996; Appeltants et al., 2001). Although T seems to increase the level of TH staining in the ventral part of this nucleus, quantitative analyses detected no statistically significant difference in the level of staining in RA between control and T-treated female canaries. T also

810

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

did not change the density of TH staining in area X. However, in contrast to RA, area X already receives a dense CAergic innervation that is similar to the surrounding lobus parolfactorius, a structure homologous to parts of the basal ganglia in mammals that is massively innervated by TH-ir fibers in both birds and mammals (Reiner et al., 1994). In two of nine T-treated birds, TH staining in area X was even slightly lower than in the surrounding tissue. This may however reflect the well-documented T-induced growth of this nucleus (Bottjer et al., 1986) resulting in the spreading out of pre-existing TH-ir fibers. We had in a previous study observed a higher intensity of TH staining in area X than in the lobus parolfactorius in gonadally intact sexually mature male and female canaries (Appeltants et al., 2001). In this study of T-treated female canaries we did not observe the male-typical pattern of TH staining in area X of the Ttreated and control females. The origin(s) of this difference cannot be ascertained with the available information. This apparent discrepancy could namely relate to minor strain differences between birds, to slightly different photoperiodical histories (light/dark cycles experienced during previous months), to the endocrine state of the subjects or to minor differences in the effectiveness of the immunocytochemical staining. Although every attempt was made to standardize these parameters, minor differences are unavoidable and they could explain the lack of specific staining in area X compared with the lobus parolfactorius. The present data nevertheless clearly indicate that T differentially affects the CAergic innervation of different song control nuclei in the female canary telencephalon. Neuroanatomical specialization of the steroidsensitive CAergic innervation of the SCS Catecholamines, the enzymes catalyzing their synthesis, and their receptors are specifically present in high concentrations in most telencephalic song control nuclei (Bottjer, 1993; Appeltants et al., 2001; Bernard and Ball, 1995; Barclay and Harding, 1988, 1990; Sakaguchi and Saito, 1989; Casto and Ball, 1994; Soha et al., 1996; Riters and Ball, 2002). These inputs arise from distinct CAergic cells groups located in the mesencephalon and rhombencephalon. HVC receives DAergic projections mainly from the mesencephalic central gray (GCt; homologous to the DAergic A11 group of mammals) and to a lesser extent from the area ventralis of Tsai (AVT; homologous to the DAergic A10 group of mammals) while the areas adjacent to HVC receive DAergic inputs mainly from the substantia nigra (SN; homologous to the DAergic A9 group of mammals) and from its caudal extension, the retroruberal nucleus (Appeltants et al., 2000). In contrast, RA receives most of its DAergic innervation in equal amounts from AVT and GCt and to a lesser extent from the SN and its caudal extension while the archistriatal tissue adjacent to RA receives CAergic inputs mainly from the SN but also from the retrorubral field, GCt and AVT (Appeltants et al., 2002a). Both HVC and RA receive also NAergic projections from the locus ceruleus complex identified as the A6 group in mammals (Appeltants et al., 2000, 2002a). Area X receives DAergic inputs from AVT (Lewis et al., 1981) but the

origins of the CAergic innervation of Nif, lMAN and mMAN are unknown at present. Although additional studies will be necessary to fully understand the complexity of these CAergic inputs to the SCS, the available results clearly suggest that the diverse and specific origin of these projections could explain the differential reactions to T treatment of the TH-ir structures located in the various song control nuclei. The methods of the present study do not allow us to determine whether T modulates DA and/or NE inputs to the telencephalic song control nuclei. Tract tracing studies have shown that DAergic as well as NAergic neurons are retrogradely labeled after injections of tracer into HVC or RA in male canaries (Appeltants et al., 2000, 2002). In male zebra finches, most telencephalic song nuclei exhibit a dense and very specific staining for TH (Bottjer, 1993). Such a pattern is not observed by immunostaining for dopamine ␤-hydroxylase, the enzyme specifically involved in the transformation of DA into NA but dopamine ␤-hydroxylase-immunoreactive fibers are nevertheless present in most song control nuclei (Mello et al., 1998). Direct assays by high performance liquid chromatography have accordingly identified high levels of DA and NA in most song control nuclei (Sakaguchi and Saito, 1989; Barclay and Harding, 1990). These data therefore indicate that both catecholamines are present in the SCS and potentially play important functional roles. This idea is also supported by the presence in these nuclei of high densities of CAergic receptors, in particular ␣2-adrenergic receptors (Riters and Ball, 2002). Additional experimental studies are needed to assess the respective roles of NA and DA in the control of singing behavior and their specific control by steroids. Endocrine specificity of the controls of CAergic inputs The T-dependent increase of the CAergic innervation of the song control nuclei could be mediated by T itself or by its androgenic and/or estrogenic metabolites. Barclay and Harding (1988, 1990) showed by direct assays of catecholamines and their turnover in the SCS of zebra finches that the hormonal modulation of the NAergic transmission is exclusively estrogen-dependent while the modulation of the DAergic transmission is frequently but not exclusively androgen-dependent. These studies were however carried out in a songbird species where song control is only marginally affected by steroids in adulthood so that these findings cannot necessarily be extrapolated to other species. Sex steroids could regulate DA and NA baseline concentrations and turnover in the song control nuclei by interacting with nuclear androgen or ERs that are present either directly in several of these nuclei (Balthazart et al., 1992; Gahr et al., 1993; Nastiuk and Clayton, 1995; Bernard et al., 1999) or at the level of the CAergic cell bodies located in the mes- and rhomb-encephalon. Recent in situ hybridization studies have indeed demonstrated the presence of sex steroid receptors at the level of GCt (mostly ARs) and AVT (ARs and ERs) in male canaries (Maney et al., 2001). The present data demonstrating increases in

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

the density of TH-ir structures in a large number of song control nuclei suggest that a significant part of the T effect on the catecholamines of the SCS results from a steroidinduced increase in transcription of TH, the rate limiting enzyme in catecholamine synthesis. These data therefore indicate that a substantial part of T effects could take place in CAergic cells bodies. They do not however rule out the possibility that some effects of T or its metabolites on the CAergic activity in the SCS result from a direct action within song control nuclei. ARs or ERs present in these nuclei could indirectly regulate the release of catecholamines by affecting the activity of their post-synaptic targets. Alternatively, steroids may also modify CAergic activity by non-genomic mechanisms. It has indeed been shown that estrogens rapidly promote the phosphorylation of TH by a direct interaction and in this way markedly increase its maximum enzymatic activity (Pasqualini et al., 1995). Changes in catecholamine synthesis can thus take place in the absence of modifications of the concentration of the synthesizing enzyme. In this respect, it must be noted that, although the increased density of TH-ir structures observed here after exposure to T strongly suggests that the steroid acted by increasing the transcription of the enzyme, alternative explanations are also possible. T could indeed affect (decrease) the turnover of TH at the level of the terminals and in this way increase its concentration or even change its tridimensional configuration (e.g. through phosphorylations, see above) and in this way alter its detection by the antibody used for immunocytochemistry. Sex steroids, catecholamines and higher order behavioral and cognitive processes The present study provides additional correlative evidence supporting the idea that CAergic inputs to the song control nuclei play specific roles in song learning and production in passerines. Functional interactions between steroids and catecholamines have been well documented in the context of brain development and sexual brain differentiation (Siddiqui and Gilmore, 1988; Reznikov and Nosenko, 1983, 1995) but also in hypothalamic areas of adult mammals and birds in relationship with the control reproductive behavior (Nock and Feder, 1981; Etgen et al., 1990, 1992). Although very few studies have addressed the question, indirect evidence strongly suggests that sex steroids and CAs should also interact in adulthood to modulate higher cognitive functions and complex information processing. Beside the well-established sex differences in cognitive abilities in mammals including human (see Kelly et al., 1999; Hampson and Kimura, 1992), several aspects of cognitive performance, that are assumed to depend on CAergic transmission, vary with changes in ovarian or testicular hormone levels in humans (Hampson, 1990; Kimura and Hampson, 1994). Furthermore, the severity and/or frequency of mental illnesses such as schizophrenia, Alzheimer’s disease, and depression that are associated to CAergic disturbance also depend on the sex or hormonal state of the subject (Flor-Henry, 1990; McNeil et al., 1984; Seeman and Lang, 1990). Many of these phe-

811

nomena may involve, at least in part, the modulatory action of sex steroids on the CAergic transmission in telencephalic brain areas important for cognition. Interestingly, it has been recently demonstrated that the CAergic innervation of prefrontal cortex that regulates complex behavioral and cognitive processes (Brozoski et al., 1979; Sawaguchi and Goldman-Rakic, 1991), is modulated by gonadal steroids (Adler et al., 1999; Kritzer and Kohama; 1998). Songbirds constitute a model that offers excellent opportunities to investigate the relationships between high levels of information processing, hormones and brain function. Despite the differences in brain organization between birds and mammals (e.g. nuclear structures in birds versus layered organization of the telencephalon in mammals), several similarities and potential homologies have been observed between the SCS and neural circuits involved in memory, cognition and use-dependent plasticity in the mammalian brain (see Bottjer and Johnson, 1997; Doupe and Khul, 1999 for discussion). This offers an opportunity to uncover invariant mechanisms by which neuromodulators such as sex steroids and catecholamines interact to modulate highly developed cognitive skills in vertebrates. Further studies will be required to identify, among the numerous effects induced by T on song behavior and its underlying neural system, which one may involve the CAergic transmission. A fraction of the morphological effects induced by T and/or its metabolites in the SCS might be mediated by DA and/or NA. For example, the increased number of neuronal soma-somatic gap junctions specifically observed in the HVC of T-treated female canaries (Gahr and Garcia-Segura, 1996) could involve the modulatory action of DA since this neuromodulator regulates gap-junctions in other models (Weiler et al., 2000). These gap-junctions could promote the synchronous firing between the HVC neurons that innervate RA (Gahr and Garcia-Segura, 1996; Dutar et al., 1998). The modulation by DA of the excitability of spiny neurons in area X has also been proposed to regulate information processing in this song nucleus and potentially modulate song behavior (Ding and Perkel, 2002). On the other hand, behavioral studies have also proposed that catecholamines, acting in the SCS, modulate different levels of information processing involved in song control. For example, disturbance of the NAergic transmission specifically impairs auditory processing of song information in female canaries (Appeltants et al., 2002b) and has been proposed to affect attentional processes related to song production in male zebra finch (Barclay et al., 1992, 1996). In the same species, local application of NA in HVC modulates the neural activity in RA which suggest that motor access to auditory feedback, which is required for song learning and maintenance, may be regulated through NAergic neuromodulation (Dave et al., 1998). The function of DA in the SCS has not been studied in great detail to this date. It has however been proposed that DA modulates processes involved in the sensory-motor integration of song and/or the amount, speed and intensity of song production (see Soha et al., 1996 and Durstewitz et al., 1999 for discussion). Interestingly song activation

812

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

following T-treatment is associated to a strong and specific increase of the CAergic innervation of the SCS that is not restricted to the motor pathway but concerns also the anterior forebrain pathway. This observation suggests that the anterior forebrain pathway could participate in the motor production of learned vocalizations in songbirds (see Doupe et al. 2000 for a discussion). Of special interest, HVC and lMAN which receive the highest densities of CAergic projections are the two song nuclei that send two massive projections to RA, the nucleus that directly and indirectly innervates the motor neurons of the syrinx. As observed in other models, CAs could stabilize neural representations within the SCS (Durstewitz et al., 2000a,b). Thus, information that is first processed within the motor and the rostral forebrain pathway and then transmitted to RA via HVC and lMAN respectively, could be amplified by catecholamines before they are transmitted and integrated within the premotor song nuclei. Whether catecholamines directly affect cognitive processes or modulate the trophic effects of T on the song system which secondarily affect song learning and production remains however to be determined and more experimental work will be needed to specify the exact functions of NA and DA in the control of song learning and expression. Acknowledgements—This research was supported by grants from the NINDS (NS-35467) to GFB and JB and grants from the Belgian FRFC (2.4555.01), the French Community of Belgium (ARC 99/04 –241), and the University of Lie`ge (Fonds Spe´ciaux pour la Recherche) to JB. DA is research fellow with the FNRS.

REFERENCES Adler A, Vescovo P, Robionson JK, Kritzer MF (1999) Gonadectomy in adult life increases tyrosine hydroxylase immunoreactivity in the prefrontal cortex and decrease open field activity in male rats. Neuroscience 89:939 –954. Appeltants D, Absil P, Balthazart J, Ball GF (2000) Identification of the origin of catecholaminergic inputs to HVC in canaries by retrograde tract tracing combined with tyrosine hydroxylase immunocytochemistry. J Chem Neuroanat 18:117–133. Appeltants D, Ball GF, Balthazart J (2001) The distribution of tyrosine hydroxylase in the canary brain: demonstration of a specific and sexually dimorphic catecholaminergic innervation of the telencephalic song control nuclei. Cell Tissue Res 304:237–259. Appeltants D, Ball GF, Balthazart J (2002a) The origin of catecholaminergic inputs to the song control nucleus RA in canaries. Neuroreport 13:649 –653. Appeltants D, Del Negro C, Balthazart J (2002b) Noradrenergic control of auditory information processing in female canaries. Behav Brain Res 133:221–235. Arnold AP (1990) The passerine bird song system as a model in neuroendocrine research. J Exp Zool 4(Suppl):22–30. Arnold AP, Nottebohm F, Plaff DW (1976) Hormone concentrating cells in vocal control and other areas of the brain of the zebra finch (Poephila guttata). J Comp Neurol 165:487–511. Bailhache T, Balthazart J (1993) The catecholaminergic system of the quail brain: immunocytochemical studies of dopamine ␤-hydroxylase and tyrosine hydroxylase. J Comp Neurol 32:230 –256. Ball GF (1994) Neurochemical specializations associated with vocal learning and production in songbirds and budgerigars. Brain Behav Evol 44:234 –246. Ball GF, Absil P, Balthazart J (1995) Peptidergic delineations of nu-

cleus interface reveal a sex difference in volume. Neuroreport 6:957–960. Ball GF, Hulse SH (1998) Bird song. Am Psychol 53:37–58. Ball GF (1999) Neuroendocrine basis of seasonal changes in vocal behavior among songbirds. In: The design of animal communication (Hauser M, Konishi M, eds), pp 213–253. Cambridge, MA: MIT Press. Ball GF, Riters LV, Balthazart J (2002) Neuroendocrinology of song behavior and avian brain plasticity: multiple sites of action of sex steroid hormones. Front Neuroendocrinol 23:137–178. Balthazart J, Foidart A, Wilson EM, Ball GF (1992) Immunocytochemical localization of androgen receptors in the male song bird and quail brain. J Comp Neurol 17:407–420. Balthazart J, Ball GF (1995) Sexual differentiation of brain and behavior in birds. Trend Endocrinol Metab 6:21–29. Barclay SR, Harding CF (1988) Androstenedione modulation of monoamine levels and turnover in hypothalamic and vocal control nuclei in the male zebra finch: steroid effects on brain monoamines. Brain Res 459:333–343. Barclay SR, Harding CF (1990) Differential modulation of monoamines levels and turnover rates by estrogen and/or androgen in hypothalamic and vocal control nuclei of male zebra finches. Brain Res 523:251–262. Barclay SR, Harding CF, Waterman SA (1992) Correlation between catecholamines levels and sexual behavior in male zebra finches. Pharmacol Biochem Behav 41:195–201. Barclay SR, Harding CF, Waterman SA (1996) Central DSP-4 treatment decreases norepinephrine levels and courtship behavior in male zebra finches. Pharmacol Biochem Behav 53:213–220. Bernard DJ, Ball GF (1995) Two histological markers reveal a similar photoperiodic differences in the volume of the high vocal center in male European starling. J Comp Neurol 360:726 –734. Bernard DJ, Bentley GE, Balthazart J, Turek FW, Ball GF (1999) Androgen receptor, estrogen receptor alpha, and estrogen receptor beta show distinct patterns of expression in forebrain song control nuclei of European starling. Endocrinology 140:4633–4643. Benton S, Nelson DA, Marler P, DeVoogd TJ (1998) Anterior forebrain pathway is needed for stable song expression in adult whitecrowned sparrows (Zonotrichia leucophrys). Behav Brain Res 96: 135–150. Beyer C, Feder HH (1987) Sex steroids and afferent input: their roles in brain sexual differentiation. Annu Rev Physiol 49:349 –364. Bottjer SW, Schoonmaker JN, Arnold AP (1986) Auditory and hormonal stimulation interact to produce neural growth in adult canaries. J Neurobiol 17:605–612. Bottjer SW, Dignan TP (1988) Joint hormonal and sensory stimulation modulate neuronal number in adult canary brains. J Neurobiol 19:624 –635. Bottjer SW, Maier E (1991) Testosterone and the incidence of hormone target cells in song-control nuclei of adult canaries. J Neurobiol 22:512–521. Bottjer SW (1993) The distribution of tyrosine hydroxylase in the brains of male and female zebra finches. J Neurobiol 24:51–69. Bottjer SW, Roselinsky H, Tran NB (1997) Sex differences in neuropeptide staining of song-control nuclei in zebra finch brains. Brain Behav Evol 50:284 –303. Bottjer SW, Johnson F (1997) Circuits, hormones, and learning: vocal behavior in songbirds. J Neurobiol 33:602–618. Brenowitz EA, Arnold AP (1990) The effects of systemic androgen treatment on androgen accumulation in song control regions of the adult female canary brain. J Neurobiol 21:837–843. Brenowitz EA, Margoliash D, Nordeen K (1997) An introduction to birdsong and the avian song system. J Neurobiol 33:495–500. Brozoski TJ, Brown RM, Rosvold HE, Goldman PS (1979) Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey. Science 205:929 –932. Canady RA, Burd GD, DeVoogd TJ, Nottebohm F (1988) Effect of testosterone on input received by an identified neuron type of the

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814 canary song system: a Golgi/electron microscopy/degeneration study. J Neurosci 8:3770 –3784. Casto JM, Ball GF (1994) Characterization and localization of D1 dopamine receptors in the sexually dimorphic vocal control nucleus, area X, and the basal ganglia of European starlings. J Neurobiol 25:767–780. Dave AS, Yu AC, Margoliash D (1998) Behavioral state modulation of auditory activity in a vocal motor system. Science 282:2250 –2254. DeVoogd TJ, Nottebohm F (1981a) Sex differences in dendritic morphology of a song control nucleus in the canary: a quantitative Golgi-study. J Comp Neurol 196:309 –316. DeVoogd TJ, Nottebohm F (1981b) Gonadal hormones induce dendritic growth in the adult brain. Science 214:202–204. DeVoogd TJ, Nixdorf B, Nottebohm F (1985) Synaptogenesis and changes in synaptic morphology related to acquisitive new behavior. Brain Res 329:304 –308. Ding L, Perkel DJ (2002) Dopamine modulates excitability of spiny neurons in the avian basal ganglia. J Neurosci 22:5210 –5218. Doupe AJ, Solis MM (1997) Song- and order-selective neurons develop in the songbird anterior forebrain during vocal learning. J Neurobiol 33:694 –709. Doupe AJ, Brainard MS, Hessler NA (2000) The song system: neural circuits essential throughout life for vocal behavior and plasticity. In: The new cognitive neurosciences, 2nd ed (Gazzaniga MS, ed), pp 451–467. Cambridge, MA: MIT Press. Doupe AJ, Kuhl PK (1999) Birdsong and human speech: common themes and mechanisms. Annu Rev Neurosci 22:567–631. Durstewitz D, Kro¨ner S, Gu¨ntu¨rku¨n O (1999) The dopaminergic innervation of the avian telencephalon. Prog Neurobiol 59:161–195. Durstewitz D, Seamans JK, Sejnowski TJ (2000) Neurocomputational models of working memory. Nat Neurosci 3 (Suppl1184 –1191. Durstewitz D, Seamans JK, Sejnowski TJ (2000b) Dopamine-mediated stabilization of delay-period activity in a network model of prefrontal cortex. J Neurophysiol 83:1733–1750. Dutar P, Vu HM, Perkel DJ (1998) Multiple cell types distinguished by physiological, pharmacological, and anatomical properties in nucleus HVC of the adult zebra finch. J Neurophysiol 80:1828 –1838. Etgen AM, Vathy I, Petitti N, Ungar S, Karkanias GB (1990) Ovarian steroids, female reproductive behavior and norepinephrine neurotransmission in the hypothalamus. In: Hormone, brain and behaviour in vertebrates, 2: behavioural activation in males and females: social interactions and reprodutive physiology: comparative physiology, Vol. 9s (Balthazart J, ed), pp 116 –128. Basel: Karger. Etgen AM, Ungar S, Petitti N (1992) Estradiol and progesterone modulation of norepinephrine neurotransmission: implications for the regulation of female reproductive behavior. J Neuroendocrinol 4:255–271. Flor-Henry P (1990) Influence of gender in schizophrenia as related to other psychopathological syndromes. Schizophr Bull 16:211–228. Foster EF, Mehta RP, Bottjer SW (1997) Axonal connections of the medial magnocellular nucleus of the anterior neostriatum in zebra finches. J Comp Neurol 382:364 –381. Gahr M, Guttinger HR, Kroodsma DE (1993) Estrogen receptors in the avian brain: survey reveals general distribution and forebrain areas unique to songbirds. J Comp Neurol 327:112–122. Gahr M, Garcia-Segura LM (1996) Testosterone-dependent increase of gap-junctions in HVC neurons of adult female canaries. Brain Res 712:69 –73. Gulledge CC, Deviche P (1999) Age- and sex-related differences in opioid receptor densities in the songbird vocal control system. J Comp Neurol 404:505–514. Hampson E (1990) Variations in sex related cognitive abilities across the menstrual cycle. Brain Cogn 14:26 –43. Hampson E, Kimura D (1992) Sex differences and hormonal influences on cognitive function in humans. In: Behavioral endocrinology (Becker JB, Breedlove SM, Crews D, eds), pp 357–398. Cambridge, MA: MIT Press.

813

Kelly SJ, Ostrowski NL, Wilson MA (1999) Gender differences in brain and behavior: hormonal and neural bases. Pharmacol Biochem Behav 64:655–664. Kimura D, Hampson E (1994) Cognitive pattern in men and women is influenced by fluctuation in sex hormones. Curr Directions Psychol Sci 3:57–61. Kritzer MF, Kohama SG (1998) Ovarian hormones influence the morphology, distribution, and density of tyrosine hydroxylase immunoreactive axons in the dorsolateral prefrontal cortex of adult rhesus monkeys. J Comp Neurol 395:1–17. Leitner S, Voigt C, Garcia-Segura LM, 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. Lewis JW, Ryan SM, Arnold AP, Butcher LL (1981) Evidence for catecholamine projection to area X in the zebra finch. J Comp Neurol 196:347–354. Maney DL, Bernard DJ, Ball GF (2001) Gonadal steroid receptor mRNA in catecholaminergic nuclei of the canary brainstem. Neurosci Lett 311:189 –192. Margoliash D (1997) Functional organization of forebrain pathways for song production and perception. J Neurobiol 33:671–693. McNeil TF, Kaij L, Malmquist-Larsson A (1984) Woman with nonorganic psychosis: pregnancy’s effect on mental health during pregnancy. Acta Psychiatr Scand 70:140 –148. Mello CV, Pinaud R, Ribeiro S (1998) Noradrenergic system of the zebra finch brain: immunocytochemical study of dopamine-␤-hydroxylase. J Comp Neurol 400:207–228. Nastiuk KL, Clayton DF (1995) The canary androgen receptor mRNA is localized in the song control nuclei in the brain and is rapidly regulated by testosterone. J Neurobiol 26:213–224. Nicholls TJ, Goldsmith AR, Dawson A (1988) Photorefractoriness in birds and comparison with mammals. Physiol Rev 68:133–176. Nock B, Feder HH (1981) Neurotransmitter modulation of steroid action in target cells that mediate reproduction and reproductive behavior. Neurosci Biobehav Rev 5:537–447. Nottebohm F, Arnold AP (1976) Sexual dimorphism in the vocal control areas in song bird brain. Science 194:211–213. Nottebohm F (1980) Testosterone triggers growth of brain vocal control nuclei in adult female canaries. Brain Res 189:429 –436. Nottebohm F, Kelley DB, Paton JA (1982) Connections of vocal control nuclei in the canary telencephalon. J Comp Neurol 207:344 –357. Nottebohm F, Nottebohm ME, Crane LA, Wingfield JC (1987) Seasonal changes in gonadal hormone levels of adult male canaries and their relation to song. Behav Neural Biol 47:197–211. Nottebohm F (1999) The anatomy and timing of vocal learning in birds. In: The design of animal communication (Hauser MD, Konishi M, eds), pp 63–110. Cambridge, MA: MIT Press. Pasqualini C, Olivier V, Guibert B, Frain O, Leviel V (1995) Acute stimulatory effect of estradiol on striatal dopamine synthesis. J Neurochem 65:1651–1657. Pesch A, Gu¨ttinger HR (1985) Der Gesang des weiblichen Kanarienvogels. J Ornithol 126:108 –110. Rasika S, Alvarez-Buylla A, Nottebohm F (1999) BDNF mediates the effects of testosterone on the survival of new neurons in an adult brain. Neuron 22:53–62. Reiner A, Karle EJ, Anderson KD, Medina L (1994) Catecholaminergic perikarya and fibers in the avian nervous system. In: Phylogeny and development of catecholamine systems in the CNS of vertebrates (Smeets WJAJ, Reiner A, eds), pp 135–181. Cambridge: Cambridge University Press. Reznikov AG, Nosenko ND (1983) It is possible that noradrenaline is the biogenic monoamine responsible for androgen-dependent sexual brain differentiation. Exp Clin Endocrinol 81:91–93. Reznikov AG, Nosenko ND (1995) Catecholamines in steroid-dependent brain development. J Steroid Biochem Mol Biol 53:349 –353.

814

D. Appeltants et al. / Neuroscience 121 (2003) 801– 814

Riters LV, Ball GF (2002) Sex differences in the densities of alpha 2-adrenergic receptors in the song control system, but not the medial preoptic nucleus in zebra finches. J Chem Neuroanat 23: 269 –277. Sakaguchi H, Saito N (1989) The acetylcholine and catecholamine contents in song control nuclei of zebra finch during song ontogeny. Dev Brain Res 47:313–317. Sawaguchi T, Goldman-Rakic PS (1991) D1 dopamine receptors in prefrontal cortex: involvement in working memory. Science 251: 947–950. Schlinger BA (1997) Sex steroids and their actions on the birdsong system. J Neurobiol 33:619 –631. Seeman MV, Lang M (1990) The role of estrogens in schizophrenia gender differences. Schizophr Bull 16:185–191. Siddiqui A, Gilmore DP (1988) Regional differences in the catechol-

amine content of the rat brain: effects of neonatal castration and androgenization. Acta Endocrinol (Copenh) 118:483–494. Soha JA, Shimizu T, Doupe AJ (1996) Development of the catecholaminergic innervation of the song system of the male zebra finch. J Neurobiol 29:473–489. Stokes TM, Leonard CM, Nottebohm F (1974) The telencephalon, diencephalon, and mesencephalon of the canary, Sirinus canaria, in stereotaxic coordinates. J Comp Neurol 156:337–374. Troyer TW, Bottjer SW (2001) Birdsong: models and mechanisms. Curr Opin Neurobiol 11:721–726. Weiler R, Pottek M, He S, Vaney DI (2000) Modulation of coupling between retinal horizontal cells by retinoic acid and endogenous dopamine. Brain Res Rev 32:121–129. Wild JM (1997) Neural pathways for the control of birdsong production. J Neurobiol 33:653–670.

(Accepted 23 June 2003)