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Gonadal steroid receptor mRNA in catecholaminergic nuclei of the canary brainstem
Neuroscience Letters 311 (2001) 189–192 www.elsevier.com/locate/neulet
Gonadal steroid receptor mRNA in catecholaminergic nuclei of the canary brainstem Donna L. Maney a,*, Daniel J. Bernard b, Gregory F. Ball a a
Behavioral Neuroendocrinology Group, Department of Psychology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA b Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208, USA Received 13 November 2000; received in revised form 30 July 2001; accepted 31 July 2001
Abstract Steroid actions in the song system may be modulated by ascending inputs from catecholaminergic (CA) brain nuclei; however, whether these nuclei contain steroid receptors is unknown. Here, we compared the distribution of androgen receptor (AR) and estrogen receptor-alpha (ER-a) mRNA with that of tyrosine hydroxylase immunoreactivity (TH-IR) in the brainstems of male canaries. Areas containing AR and ER-a mRNA overlapped with areas containing TH-IR cell bodies in the locus ceruleus and the area ventralis of Tsai. The substantia nigra and the midbrain central gray contained both TH-IR and AR mRNA. The presence of AR and ER-a within CA cell groups suggests that sex steroid hormones may modulate song production at the site of CA synthesis. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Androgen receptor; Estrogen receptor; Tyrosine hydroxylase; Area ventralis; Central gray; Locus ceruleus; Song
Singing behavior is controlled by a series of well-defined brain nuclei known as the song control system (Fig. 1). Many of these nuclei receive heavy innervation from catecholaminergic (CA) fibers. For example, autoradiographic studies [4,9] indicate dense CA binding in HVc (sometimes called the high vocal center) and in the robust nucleus of the archistriatum (RA), which are part of a descending motor pathway innervating the syrinx, and in Area X, which is considered to be a portion of the avian basal ganglia. The same nuclei also show dense immunolabeling for fiber terminals containing tyrosine hydroxylase (TH), an enzyme involved in CA synthesis [11,20]. CA cell groups are implicated in singing behavior; reducing brain norepinephrine levels delays the onset of singing and reduces the number of songs during courtship in male zebra finches [8]. CA inputs to the song control system are modulated by gonadal steroids. For example, manipulation of plasma androgen and estrogen levels affects the rates of CA turnover in many song control nuclei [6]. Estrogens regulate the noradrenergic inputs to the song system, whereas dopaminergic inputs are affected by both androgens and estrogens [6]. In addition, seasonal differences in plasma sex steroid * Corresponding author. Tel.: 11-410-516-6007; fax: 11-410516-6205. E-mail address: [email protected] (D.L. Maney).
levels appear to mediate seasonal changes in CA innervation – the boundaries of HVc, as defined by alpha2-adrenergic receptor density, change according to reproductive condition in European starlings (Sturnus vulgaris) [9]. CA pathways may play a key role in mediating the effects of steroids on reproductive behaviors. But where do gonadal steroids act within CA systems? Androgens and estrogens most likely exert their effects on CA innervation and release by binding to receptors at either the source of CA input i.e. the cell bodies in the brainstem, or at the targets in the song system, or both (Fig. 1). There is already a large body of research describing the distribution of gonadal hormone receptors in the song control system; for example, androgen receptors (ARs) are found in HVc, RA, and other song nuclei [2,10,17]. Estrogen receptors are not as highly localized to the song system but can be found in HVc [10,12]. Thus, steroid hormones could affect CA function by acting at the targets of CA innervation. Alternatively, steroids may exert their effects at the source of CA input. Studies in zebra finches (Taeniopygia guttata) and canaries (Serinus canaria) suggest that telencephalic CA innervation originates primarily from two cell groups in the mesencephalon and one in the rhombencephalon. In the mesencephalon, the area ventralis of Tsai (AVT), thought to be homologous to the mammalian ventral tegmental area (VTA) [11], and the midbrain central gray
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 02 15 7- 7
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Fig. 1. Schematic drawing in the sagittal plane of the known CA inputs to the song control system. Steroids could act at steroid receptors in the song system (white), and/or at the sources of CA input (black). HVc, high vocal center; RA, nucleus robustus archistriatalis; lMAN, lateral magnocellular nucleus of the anterior neostriatum; nXII, nucleus of the hypoglossal nerve; AVT, area ventralis of Tsai; GCt, substantia grisea centralis; LoC, locus ceruleus.
(GCt) send TH-immunopositive fibers to HVc [1]. AVT also sends a CA projection to Area X [14]. In the rhombencephalon, the locus ceruleus (LoC) sends a noradrenergic projection to HVc [1]. RA receives input from AVT and from LoC [15], but it is not known whether these afferents are catecholaminergic. Homologous CA cells in the mammalian brainstem concentrate gonadal steroids [16] and contain gonadal steroid receptors [13]. Here, we compare the distribution of AR and estrogen receptoralpha (ER-a) mRNA with TH immunoreactivity (TH-IR) in adjacent sections of canary brainstem in order to determine whether CA cell groups express gonadal steroid receptors in this species. 6 male canaries obtained from local breeders were killed by rapid decapitation and their brains immediately frozen on dry ice. Four sets of 25 mm sections were cut on a cryostat such that sections in each set were 100 mm apart. One set was processed for in situ hybridization for AR mRNA, one set for in situ hybridization for ER-a mRNA, one for immunocytochemistry (ICC) for TH, and one was stained with thionin. In situ: this procedure was carried out as previously described [10]. Adjacent sections were hybridized with antisense riboprobes directed against starling ER-a or AR mRNA. Slides were exposed to Biomax film for 5–9 days before developing. ICC: slides were air-dried for 10–15 min and fixed for 1 min in 4% paraformaldehyde, washed in PBS, and incubated in 0.3% H2O2 in PBS for 30 min. After a wash in PBS, they were incubated in 10% normal horse serum (Vector; Burlingame, CA, USA) in PBS with 0.3% Triton
X-100 (0.3% PBST) for 1 h at room temperature, then overnight at 48C. They were then incubated with mouse monoclonal anti-TH antibodies (Diasorin, Stillwater, MN, USA) diluted 1:2000 in PBST at 48C for 3 days. The specificity of this antibody for use in avian species has been verified elsewhere [3]. Sections were washed in 0.1% PBST and incubated for 1 h in horse anti-mouse IgG (Vector), diluted 1:200 in 0.3% PBST. After a wash in 0.1% PBST, they were incubated in avidin-biotin horseradish peroxidase complex (Vectastain ABC, Elite Kit; Vector) diluted 1:200 for 1 h. They were washed once more in 0.1% PBST, and TH-IR was visualized using SigmaFast DAB tablets (Sigma, St. Louis, MO, USA). Slides were then washed extensively in PBS, dehydrated, and coverslipped in DPX (Aldrich, Milwaukee, WI, USA). In the brainstem, AR mRNA labeling above background was visible in the caudal part of the AVT (Fig. 2D), the substantia nigra (SN; Fig. 2D,G), the GCt (Fig. 2G), and the LoC (Fig. 2J), These areas correspond to CA cell groups A10 (AVT), A9 (SN), A11 (GCt), and A6 (LoC) [18]. Other AR labeling was consistent with that previously reported in this species [2,10,17]: the median eminence, tuberal hypothalamus, HVc, RA (all not shown), nucleus intercollicularis (ICo; Fig. 2D,G) all labeled heavily. Whereas the caudal part of AVT, not the rostral part, expressed AR mRNA (Fig. 2A,D), ER-a mRNA labeling was confined to the rostral part of this nucleus (A10; Fig. 2C,F). No ER-a mRNA expression was visible in the SN or the GCt (Fig. 2F,I), but it was readily apparent in the LoC (A6; Fig. 2L), which labeled more heavily for ER-a than AR mRNA. Other ER-a expression was consistent with that previously reported [10,12]: dense labeling in the median eminence and tuberal hypothalamus and moderate labeling in HVc (not shown). TH-IR was consistent with that previously reported in this species [5]. In the areas relevant to this study, TH-IR cell bodies and dense fiber networks were found in both rostral and caudal AVT (A10; Fig. 2B,E), SN (A9; Fig. 2E,H), GCt (A11; Fig. 2H), and LoC (A6; Fig. 2K). Our results are consistent with the hypothesis that steroids act at the site of CA synthesis. Steroids could, therefore, act at both the sources and targets of the CA system (Fig. 1). The CA cell groups that contain steroid receptor mRNA are known to project to the song system [1], which itself contains steroid receptors [2,10,17]. Acting at the site of CA synthesis, gonadal steroids could regulate production of CA synthetic enzymes such as TH or dopamine beta hydroxylase. Within the song control system, steroid hormone receptors could be located presynaptically, on within the axon terminals of CA afferents. Acting here or at the cell bodies, steroid hormones may mediate changes in the pattern or number of CA axons terminating in the song control system. Alternatively, gonadal hormone receptors could be located postsynaptically, in the cells of the song control system itself–perhaps altering the expression of alpha2-adrenergic receptors.
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Fig. 2. Video images of films showing AR mRNA distribution (A,D,G,J), ER-a mRNA distribution (C,F,I,L); and slides showing TH-IR (B,E,H,K). Care was taken to present consecutive sections from the same animal, and as a result the images shown do not represent the best labeling in every case. Across each horizontal row, sections are consecutive (25 mm apart). Arrows point to regions of TH-IR that overlap areas of gonadal steroid receptor mRNA labeling. Note AR mRNA labeling above background in caudal AVT (D) and light labeling of rostral SN (D). We do not consider AR mRNA labeling in rostral AVT (A) or ER-a mRNA labeling in caudal AVT or SN (F) to be reliably above background. AR mRNA labeling is present in GCt and SN (G), and in LoC (J). Note also ER-a mRNA labeling above background in rostral AVT (C), and LoC (L) but not GCt or SN (I). All images here came from a single bird, but films from all other birds confirmed this pattern of labeling. See Fig. 1 for abbreviation key.
AR and ER-a mRNA were differentially distributed within AVT. AR mRNA was limited to the caudal part of the nucleus, whereas ER-a mRNA was found primarily in the rostral part (Fig. 2A–F). This pattern of labeling was confirmed for all subjects. Kritzer [13] reported that ER mRNA in the VTA is generally restricted to the ventral (paranigral) region, whereas AR mRNA is more widespread, appearing in the dorsal (parabrachial) as well as the ventral regions. It is possible that the caudal and rostral portions of the avian AVT project to slightly different parts of the song system, such that androgens and estrogens may affect CA activity differentially in different regions. AVT sends a dopaminergic projection to Area X [14], a song control nucleus that, although it does not contain
steroid receptors [10], contains steroid-sensitive CA inputs [7]. In GCt, the primary source of CA input to HVc [1], AR mRNA labeling was more abundant than labeling for ER-a mRNA. (Fig. 2G–I). Note that Gahr et al. [12] reported ER immunoreactivity in GCt; ER-a mRNA is perhaps present but not readily detectable by our methods here. We also found that SN, which is contiguous with the caudal extent of AVT, contains detectable AR but not ER-a mRNA (Fig. 2G–I; see also light labeling of rostral SN in Fig. 2D). SN may send a weak projection to HVc but is not a major source of CA input [1]. In this study, only the a subtype of the ER was localized. The other known subtype, ER-b, is not detectable in any of
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the telencephalic song control nuclei in starlings [10]. Preliminary studies in starlings failed to localize ER-b mRNA in the brainstem. However, the mammalian literature shows its distribution in the SN, the VTA, and the LoC [19]. Future studies in the canary will attempt co-localize ER-b in brainstem CA neurons. In summary, we compared the distribution of AR and ERa mRNA with TH-IR in the brainstem of male canaries. Three TH-IR cell groups known to project to the song system [1], LoC, GCt, and AVT, were found to contain AR mRNA expressing cells. In addition, AR mRNA was visualized in the SN, another TH-IR cell group. ER-a mRNA was detected in AVT and LoC. We did not co-localize the TH-IR and steroid receptor mRNA in the same cells; however, studies in mammals have shown that CA cells in homologous nuclei do contain both AR and ER [13,16]. It is likely, then, that future co-localization studies in birds will show gonadal steroid receptor expression within TH cells. We thank Brian Spar for administrative assistance and Jacques Balthazart and an anonymous reviewer for thoughtful comments on the manuscript. This research was supported by grants from the National Science Foundation (DBN 9804129) and the National Institutes of Health (RO1 NS 35467). [1] Appeltants, D., Absil, P., Balthazart, J. and Ball, G.F., Identification of the origin of catecholaminergic inputs to HVc in canaries by retrograde tract tracing combined with tyrosine hydroxylase immunocytochemistry, J. Chem. Neuroanat., 18 (2000) 117–133. [2] Arnold, A.P., Nottebohm, F. and Pfaff, D.W., Hormone concentrating cells in vocal control and other areas of the brain of the zebra finch (Poephila guttata), J. Comp. Neurol., 165 (1976) 487–511. [3] Bailhache, T. and Balthazart, J., Effects of neurochemical lesions of the preoptic area on male sexual behavior in the Japanese quail, J. Comp. Neurol., 329 (1993) 230–256. [4] Ball, G.F., Neurochemical specializations associated with vocal learning and production in songbirds and budgerigars, Brain Behav. Evol., 44 (1994) 234–246. [5] Balthazart, J. and Ball, G.F., Identification of catecholaminergic cell groups in the brainstem of the canary, zebra finch, white-throated sparrow, and budgerigar by tyrosine hydroxylase immunocytochemistry, Belg. J. Zool., 126 (1996) 65–78. [6] Barclay, S.R. and Harding, C.F., Differential modulation of monoamine levels and turnover rates by estrogen and/or androgen in hypothalamic and vocal control nuclei of male zebra finches, Brain Res., 523 (1990) 251–262. [7] Barclay, S.R. and Harding, C.F., Androstenedione modula-
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tion of monoamine levels and turnover in hypothalamic and vocal control nuclei in the male zebra finch: steroid effects on brain monoamines, Brain Res., 459 (1988) 333– 343. Barclay, S.R., Harding, C.F. and Waterman, S.A., Central DSP-4 treatment decreases norepinephrine levels and courtship behavior in male zebra finches, Pharmacol. Biochem. Behav., 53 (1996) 213–220. Bernard, D.J. and Ball, G.F., Two histological markers reveal a similar photoperiodic difference in the volume of the high vocal center in male European starlings, J. Comp. Neurol., 360 (1995) 726–734. Bernard, D.J., Bentley, G.E., Balthazart, J., Turek, F.W. and Ball, G.F., Androgen receptor, estrogen receptor alpha, and estrogen receptor beta show distinct patterns of expression in forebrain song control nuclei of European starlings, Endocrinology, 140 (1999) 4633–4643. Bottjer, S.W., The distribution of tyrosine hydroxylase immunoreactivity in the brains of male and female zebra finches, J. Neurobiol., 24 (1993) 51–69. Gahr, M., Guttinger, H.R. and Kroodsma, D.E., Estrogen receptors in the avian brain: survey reveals general distribution and forebrain areas unique to songbirds, J. Comp. Neurol., 327 (1993) 112–122. Kritzer, M.F., Selective colocalization of immunoreactivity for intracellular gonadal hormone receptors and tyrosine hydroxylase in the ventral tegmental area, substantia nigra, and retrorubral fields in the rat, J. Comp. Neurol, 379 (1997) 247–260. Lewis, J.W., Ryan, S.M., Arnold, A.P. and Butcher, L.L., Evidence for a catecholaminergic projection to area X in the zebra finch, J. Comp. Neurol., 196 (1981) 347–354. Li, R. and Sakaguchi, H., Cholinergic innervation of the song control nuclei by the ventral paleostriatum in the zebra finch: a double-labeling study with retrograde fluorescent tracers and choline acetyltransferase immunohistochemistry, Brain Res., 763 (1997) 239–246. Heritage, A.S., Stumpf, W.E., Sar, M. and Grant, L.D., Brainstem catecholamine neurons are target sites for sex steroid hormones, Science, 207 (1980) 1377–1379. Nastiuk, K.L. and Clayton, D.F., The canary androgen receptor mRNA is localized in the song control nuclei of the brain and is rapidly regulated by testosterone, J. Neurobiol., 26 (1995) 213–224. Reiner, A., Karle, E.J., Anderson, K.D. and Medina, L., Catecholaminergic perikarya and fibers in the avian nervous system, In W.J.A.J. Smeets and A. Reiners (Eds.), Phylogeny and Development of Catecholamine Systems in the CNS, Cambridge University Press, Cambridge, 1994, pp. 135–181. Shughrue, P.J., Scrimo, P.J. and Merchenthaler, I., Estrogen binding and estrogen receptor characterization (ERalpha and ERbeta) in the cholinergic neurons of the rat basal forebrain, Neuroscience, 96 (2000) 41–49. Soha, J.A., Shimizu, T. and Doupe, A.J., Development of the catecholaminergic innervation of the song system of the male zebra finch, J. Neurobiol., 29 (1996) 473–489.
Gonadal steroid receptor mRNA in catecholaminergic nuclei of the canary brainstem Donna L. Maney a,*, Daniel J. Bernard b, Gregory F. Ball a a
Behavioral Neuroendocrinology Group, Department of Psychology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA b Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208, USA Received 13 November 2000; received in revised form 30 July 2001; accepted 31 July 2001
Abstract Steroid actions in the song system may be modulated by ascending inputs from catecholaminergic (CA) brain nuclei; however, whether these nuclei contain steroid receptors is unknown. Here, we compared the distribution of androgen receptor (AR) and estrogen receptor-alpha (ER-a) mRNA with that of tyrosine hydroxylase immunoreactivity (TH-IR) in the brainstems of male canaries. Areas containing AR and ER-a mRNA overlapped with areas containing TH-IR cell bodies in the locus ceruleus and the area ventralis of Tsai. The substantia nigra and the midbrain central gray contained both TH-IR and AR mRNA. The presence of AR and ER-a within CA cell groups suggests that sex steroid hormones may modulate song production at the site of CA synthesis. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Androgen receptor; Estrogen receptor; Tyrosine hydroxylase; Area ventralis; Central gray; Locus ceruleus; Song
Singing behavior is controlled by a series of well-defined brain nuclei known as the song control system (Fig. 1). Many of these nuclei receive heavy innervation from catecholaminergic (CA) fibers. For example, autoradiographic studies [4,9] indicate dense CA binding in HVc (sometimes called the high vocal center) and in the robust nucleus of the archistriatum (RA), which are part of a descending motor pathway innervating the syrinx, and in Area X, which is considered to be a portion of the avian basal ganglia. The same nuclei also show dense immunolabeling for fiber terminals containing tyrosine hydroxylase (TH), an enzyme involved in CA synthesis [11,20]. CA cell groups are implicated in singing behavior; reducing brain norepinephrine levels delays the onset of singing and reduces the number of songs during courtship in male zebra finches [8]. CA inputs to the song control system are modulated by gonadal steroids. For example, manipulation of plasma androgen and estrogen levels affects the rates of CA turnover in many song control nuclei [6]. Estrogens regulate the noradrenergic inputs to the song system, whereas dopaminergic inputs are affected by both androgens and estrogens [6]. In addition, seasonal differences in plasma sex steroid * Corresponding author. Tel.: 11-410-516-6007; fax: 11-410516-6205. E-mail address: [email protected] (D.L. Maney).
levels appear to mediate seasonal changes in CA innervation – the boundaries of HVc, as defined by alpha2-adrenergic receptor density, change according to reproductive condition in European starlings (Sturnus vulgaris) [9]. CA pathways may play a key role in mediating the effects of steroids on reproductive behaviors. But where do gonadal steroids act within CA systems? Androgens and estrogens most likely exert their effects on CA innervation and release by binding to receptors at either the source of CA input i.e. the cell bodies in the brainstem, or at the targets in the song system, or both (Fig. 1). There is already a large body of research describing the distribution of gonadal hormone receptors in the song control system; for example, androgen receptors (ARs) are found in HVc, RA, and other song nuclei [2,10,17]. Estrogen receptors are not as highly localized to the song system but can be found in HVc [10,12]. Thus, steroid hormones could affect CA function by acting at the targets of CA innervation. Alternatively, steroids may exert their effects at the source of CA input. Studies in zebra finches (Taeniopygia guttata) and canaries (Serinus canaria) suggest that telencephalic CA innervation originates primarily from two cell groups in the mesencephalon and one in the rhombencephalon. In the mesencephalon, the area ventralis of Tsai (AVT), thought to be homologous to the mammalian ventral tegmental area (VTA) [11], and the midbrain central gray
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 02 15 7- 7
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Fig. 1. Schematic drawing in the sagittal plane of the known CA inputs to the song control system. Steroids could act at steroid receptors in the song system (white), and/or at the sources of CA input (black). HVc, high vocal center; RA, nucleus robustus archistriatalis; lMAN, lateral magnocellular nucleus of the anterior neostriatum; nXII, nucleus of the hypoglossal nerve; AVT, area ventralis of Tsai; GCt, substantia grisea centralis; LoC, locus ceruleus.
(GCt) send TH-immunopositive fibers to HVc [1]. AVT also sends a CA projection to Area X [14]. In the rhombencephalon, the locus ceruleus (LoC) sends a noradrenergic projection to HVc [1]. RA receives input from AVT and from LoC [15], but it is not known whether these afferents are catecholaminergic. Homologous CA cells in the mammalian brainstem concentrate gonadal steroids [16] and contain gonadal steroid receptors [13]. Here, we compare the distribution of AR and estrogen receptoralpha (ER-a) mRNA with TH immunoreactivity (TH-IR) in adjacent sections of canary brainstem in order to determine whether CA cell groups express gonadal steroid receptors in this species. 6 male canaries obtained from local breeders were killed by rapid decapitation and their brains immediately frozen on dry ice. Four sets of 25 mm sections were cut on a cryostat such that sections in each set were 100 mm apart. One set was processed for in situ hybridization for AR mRNA, one set for in situ hybridization for ER-a mRNA, one for immunocytochemistry (ICC) for TH, and one was stained with thionin. In situ: this procedure was carried out as previously described [10]. Adjacent sections were hybridized with antisense riboprobes directed against starling ER-a or AR mRNA. Slides were exposed to Biomax film for 5–9 days before developing. ICC: slides were air-dried for 10–15 min and fixed for 1 min in 4% paraformaldehyde, washed in PBS, and incubated in 0.3% H2O2 in PBS for 30 min. After a wash in PBS, they were incubated in 10% normal horse serum (Vector; Burlingame, CA, USA) in PBS with 0.3% Triton
X-100 (0.3% PBST) for 1 h at room temperature, then overnight at 48C. They were then incubated with mouse monoclonal anti-TH antibodies (Diasorin, Stillwater, MN, USA) diluted 1:2000 in PBST at 48C for 3 days. The specificity of this antibody for use in avian species has been verified elsewhere [3]. Sections were washed in 0.1% PBST and incubated for 1 h in horse anti-mouse IgG (Vector), diluted 1:200 in 0.3% PBST. After a wash in 0.1% PBST, they were incubated in avidin-biotin horseradish peroxidase complex (Vectastain ABC, Elite Kit; Vector) diluted 1:200 for 1 h. They were washed once more in 0.1% PBST, and TH-IR was visualized using SigmaFast DAB tablets (Sigma, St. Louis, MO, USA). Slides were then washed extensively in PBS, dehydrated, and coverslipped in DPX (Aldrich, Milwaukee, WI, USA). In the brainstem, AR mRNA labeling above background was visible in the caudal part of the AVT (Fig. 2D), the substantia nigra (SN; Fig. 2D,G), the GCt (Fig. 2G), and the LoC (Fig. 2J), These areas correspond to CA cell groups A10 (AVT), A9 (SN), A11 (GCt), and A6 (LoC) [18]. Other AR labeling was consistent with that previously reported in this species [2,10,17]: the median eminence, tuberal hypothalamus, HVc, RA (all not shown), nucleus intercollicularis (ICo; Fig. 2D,G) all labeled heavily. Whereas the caudal part of AVT, not the rostral part, expressed AR mRNA (Fig. 2A,D), ER-a mRNA labeling was confined to the rostral part of this nucleus (A10; Fig. 2C,F). No ER-a mRNA expression was visible in the SN or the GCt (Fig. 2F,I), but it was readily apparent in the LoC (A6; Fig. 2L), which labeled more heavily for ER-a than AR mRNA. Other ER-a expression was consistent with that previously reported [10,12]: dense labeling in the median eminence and tuberal hypothalamus and moderate labeling in HVc (not shown). TH-IR was consistent with that previously reported in this species [5]. In the areas relevant to this study, TH-IR cell bodies and dense fiber networks were found in both rostral and caudal AVT (A10; Fig. 2B,E), SN (A9; Fig. 2E,H), GCt (A11; Fig. 2H), and LoC (A6; Fig. 2K). Our results are consistent with the hypothesis that steroids act at the site of CA synthesis. Steroids could, therefore, act at both the sources and targets of the CA system (Fig. 1). The CA cell groups that contain steroid receptor mRNA are known to project to the song system [1], which itself contains steroid receptors [2,10,17]. Acting at the site of CA synthesis, gonadal steroids could regulate production of CA synthetic enzymes such as TH or dopamine beta hydroxylase. Within the song control system, steroid hormone receptors could be located presynaptically, on within the axon terminals of CA afferents. Acting here or at the cell bodies, steroid hormones may mediate changes in the pattern or number of CA axons terminating in the song control system. Alternatively, gonadal hormone receptors could be located postsynaptically, in the cells of the song control system itself–perhaps altering the expression of alpha2-adrenergic receptors.
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Fig. 2. Video images of films showing AR mRNA distribution (A,D,G,J), ER-a mRNA distribution (C,F,I,L); and slides showing TH-IR (B,E,H,K). Care was taken to present consecutive sections from the same animal, and as a result the images shown do not represent the best labeling in every case. Across each horizontal row, sections are consecutive (25 mm apart). Arrows point to regions of TH-IR that overlap areas of gonadal steroid receptor mRNA labeling. Note AR mRNA labeling above background in caudal AVT (D) and light labeling of rostral SN (D). We do not consider AR mRNA labeling in rostral AVT (A) or ER-a mRNA labeling in caudal AVT or SN (F) to be reliably above background. AR mRNA labeling is present in GCt and SN (G), and in LoC (J). Note also ER-a mRNA labeling above background in rostral AVT (C), and LoC (L) but not GCt or SN (I). All images here came from a single bird, but films from all other birds confirmed this pattern of labeling. See Fig. 1 for abbreviation key.
AR and ER-a mRNA were differentially distributed within AVT. AR mRNA was limited to the caudal part of the nucleus, whereas ER-a mRNA was found primarily in the rostral part (Fig. 2A–F). This pattern of labeling was confirmed for all subjects. Kritzer [13] reported that ER mRNA in the VTA is generally restricted to the ventral (paranigral) region, whereas AR mRNA is more widespread, appearing in the dorsal (parabrachial) as well as the ventral regions. It is possible that the caudal and rostral portions of the avian AVT project to slightly different parts of the song system, such that androgens and estrogens may affect CA activity differentially in different regions. AVT sends a dopaminergic projection to Area X [14], a song control nucleus that, although it does not contain
steroid receptors [10], contains steroid-sensitive CA inputs [7]. In GCt, the primary source of CA input to HVc [1], AR mRNA labeling was more abundant than labeling for ER-a mRNA. (Fig. 2G–I). Note that Gahr et al. [12] reported ER immunoreactivity in GCt; ER-a mRNA is perhaps present but not readily detectable by our methods here. We also found that SN, which is contiguous with the caudal extent of AVT, contains detectable AR but not ER-a mRNA (Fig. 2G–I; see also light labeling of rostral SN in Fig. 2D). SN may send a weak projection to HVc but is not a major source of CA input [1]. In this study, only the a subtype of the ER was localized. The other known subtype, ER-b, is not detectable in any of
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the telencephalic song control nuclei in starlings [10]. Preliminary studies in starlings failed to localize ER-b mRNA in the brainstem. However, the mammalian literature shows its distribution in the SN, the VTA, and the LoC [19]. Future studies in the canary will attempt co-localize ER-b in brainstem CA neurons. In summary, we compared the distribution of AR and ERa mRNA with TH-IR in the brainstem of male canaries. Three TH-IR cell groups known to project to the song system [1], LoC, GCt, and AVT, were found to contain AR mRNA expressing cells. In addition, AR mRNA was visualized in the SN, another TH-IR cell group. ER-a mRNA was detected in AVT and LoC. We did not co-localize the TH-IR and steroid receptor mRNA in the same cells; however, studies in mammals have shown that CA cells in homologous nuclei do contain both AR and ER [13,16]. It is likely, then, that future co-localization studies in birds will show gonadal steroid receptor expression within TH cells. We thank Brian Spar for administrative assistance and Jacques Balthazart and an anonymous reviewer for thoughtful comments on the manuscript. This research was supported by grants from the National Science Foundation (DBN 9804129) and the National Institutes of Health (RO1 NS 35467). [1] Appeltants, D., Absil, P., Balthazart, J. and Ball, G.F., Identification of the origin of catecholaminergic inputs to HVc in canaries by retrograde tract tracing combined with tyrosine hydroxylase immunocytochemistry, J. Chem. Neuroanat., 18 (2000) 117–133. [2] Arnold, A.P., Nottebohm, F. and Pfaff, D.W., Hormone concentrating cells in vocal control and other areas of the brain of the zebra finch (Poephila guttata), J. Comp. Neurol., 165 (1976) 487–511. [3] Bailhache, T. and Balthazart, J., Effects of neurochemical lesions of the preoptic area on male sexual behavior in the Japanese quail, J. Comp. Neurol., 329 (1993) 230–256. [4] Ball, G.F., Neurochemical specializations associated with vocal learning and production in songbirds and budgerigars, Brain Behav. Evol., 44 (1994) 234–246. [5] Balthazart, J. and Ball, G.F., Identification of catecholaminergic cell groups in the brainstem of the canary, zebra finch, white-throated sparrow, and budgerigar by tyrosine hydroxylase immunocytochemistry, Belg. J. Zool., 126 (1996) 65–78. [6] Barclay, S.R. and Harding, C.F., Differential modulation of monoamine levels and turnover rates by estrogen and/or androgen in hypothalamic and vocal control nuclei of male zebra finches, Brain Res., 523 (1990) 251–262. [7] Barclay, S.R. and Harding, C.F., Androstenedione modula-
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