Article Figures & Data
Figures
- Figure 1.
Steroid hormone receptors are expressed throughout the vocal motor pathway of the song control circuit. This simplified schematic shows the distribution of ARs, ERs, and aromatase (AROM), the enzyme that converts T to estradiol, in the vocal motor pathway (VMP) of the song control system. HVC (proper name) projects to RA. RA controls motor neurons in the tracheosyringeal portion of the twelfth cranial nerve (nXIIts), which in turn controls the muscles of the syrinx, the avian vocal organ, to generate birdsong. ARs are expressed at every level of the VMP, whereas ERs are expressed only in HVC. *ER is expressed in HVC only in some species, including canaries (Ball et al., 2002). **AROM mRNA is not found in HVC, but AROM protein has been observed in presynaptic boutons in HVC in zebra finches (Peterson et al., 2005).
- Figure 2.
Flutamide specificity and syllable of interest for analysis of bandwidth stereotypy. A, The white arrow indicates an example of the syllable used in the automated syllable analysis. B, Effects of flutamide-filled implants targeting the area directly adjacent to HVC were correlated to their distance medial to the lateral part of HVC (lHVC). All birds with flutamide-filled implants presented here were in the “Flut” group as their cannula tip was in each case near HVC. C, Photomicrograph of Nissl-stained section illustrating an implant located near HVC (delineated by dashed border). D, Flutamide-filled implants targeting RA were also highly spatially effective depending on their distance dorsal to the dorsal edge of RA (dRA). E, Photomicrograph of Nissl-stained section illustrating an implant located near RA (delineated by dashed border). F, CP was not different in birds treated with empty cannula (Ctrl) or flutamide-filled cannula (Flut), suggesting flutamide did not leak into the general circulation (Tramontin et al., 2003; Alward et al., 2016c). Scale bars: C, E, 200 μm.
- Figure 3.
Androgen receptor antagonism in HVC bilaterally decreases whole-song bandwidth stereotypy. A, B, Examples of canary whole songs on day 21 from birds treated bilaterally with control (Ctrl) or flutamide-filled (Flut) cannula targeting HVC. Scale bar, 1 s. C, By day 21, AR blockade in HVC led to an increase in whole-song bandwidth CV, a measure of song stereotypy (the higher the CV, the lower the stereotypy). Symbols represent mean ± SEM. *p < 0.05 compared with Ctrl on day 21. D, E, Histograms show the highly variable distribution of the proportion of whole songs sung at different bandwidths for birds with AR blocked in HVC (blue) or controls (gray) on day 21.
- Figure 4.
Androgen receptor antagonism in RA bilaterally decreases whole-song bandwidth stereotypy. A, B, Examples of canary whole songs on day 21 from birds treated bilaterally with control (Ctrl) or flutamide-filled (Flut) cannula targeting RA. Scale bar, 1 s. C, Androgen receptor blockade in RA led to a rapid, large increase whole-song bandwidth CV, a measure of song stereotypy (the higher the CV, the lower the stereotypy). Symbols represent mean ± SEM. **At the top of a graph indicates p = 0.01 for the overall effect of treatment. D, E, Histograms show the highly variable distribution of the proportion of whole songs sung at different bandwidths for birds with androgen receptors blocked in RA (red) or controls (dark gray) on day 21.
- Figure 5.
Androgen signaling in RA but not in HVC controls bandwidth stereotypy of syllables and trills. There were no differences between birds treated with flutamide-filled cannula in HVC (Flut) and controls (Ctrl) in terms of trill (A) and syllable (B) bandwidth CV, a measure of stereotypy (higher CV, lower stereotypy). C, D, AR blockade in RA led to an increase in trill and syllable bandwidth CV. Symbols represent mean ± SEM. ***At the top of a graph indicates p < 0.01 for the overall effect of treatment; **p = 0.01 for an overall effect of treatment. E–G, Three successive trills sung by a control bird showing high stereotypy (i.e., low variability) from trill to trill as well as from syllable to syllable, compared with trills in birds with AR blocked in RA (H–J), which show lower stereotypy (i.e., more variability) from trill to trill and syllable to syllable. K–M, All measures of stereotypy correlated significantly with one another in birds treated with flutamide near RA and their controls. Symbols for individual birds are color-coded as in C, D.
- Figure 6.
Androgen receptors in HVC control syllable sequence variability and syllable-type usage. A, Blocking ARs in HVC led birds to sing with enhanced syllable sequence variability (first-order entropy; see Materials and Methods). B, C, Birds treated with flutamide in HVC (Flut) showed more random (i.e., variable) transitions from their dominant syllable type compared with controls (Ctrl). D, By day 21, antagonism of AR in HVC also caused birds to sing with high syllable-type usage variability (zero-order entropy; see Materials and Methods). E, F, Pie charts showing the proportion of use of different syllables (each slice = different syllable). Use of different syllables was more equally distributed (i.e., closer to a random distribution) in flutamide birds compared with control birds. G–I, Syllable-type usage variability correlated positively with whole-song bandwidth CV (the higher the bandwidth CV, the less stereotypic) only on day 21 (I) but not on the two previous time points (G, H), which is when birds with their AR in HVC blocked sang with significantly more whole-song bandwidth CV compared with controls (Fig. 2C–E). There were no differences between birds treated with flutamide-filled cannula in RA (Flut) and controls (Ctrl) in terms of syllable sequence variability (J) or syllable-type usage variability (K; first-order and zero-order entropy; see Materials and Methods). In A and D, symbols represent mean ± SEM. ***At the top of a graph indicates p < 0.01 for the overall effect of treatment in A; *p < 0.05 comparing Flut to Ctrl on day 21 in B. G–I, Symbols for individual birds are color-coded as in A, B.
- Figure 7.
Blocking androgen receptors in HVC causes birds to sing trills of longer duration, whereas blockade of androgen receptors in RA extends song duration by causing birds to sing more syllables per song. A, Androgen signaling in HVC does not appear to control whole-song duration; (B) however, blockade of androgen signaling in HVC with flutamide (Flut) leads birds to produce trills of longer duration compared with controls (Ctrl). C, Birds with their androgen receptors in RA blocked (Flut) sang longer songs than controls (Ctrl), (D, E) an effect driven by these birds singing more syllables per song. Bars represent mean ± SEM. *p < 0.05 for an effect of treatment. Symbols in C for individual birds are color-coded like as A, B.
- Figure 8.
Working model of the regulation of birdsong by androgen signaling. This model posits that increased androgens and estrogens (e.g., during the breeding season) act at multiple sites in the songbird brain to regulate distinct features of birdsong by modulating neurophysiological features. Androgens act in an anatomically distinct nonredundant manner to regulate specific features of birdsong, but these effects are also modulated by the transsynaptic feedback between these nuclei. Previous studies indicate androgens and estrogens acting within the POM may regulate the motivation to sing (Alward et al., 2013, 2016c). The POM likely influences the song control system indirectly, via projections to the ventral tegmental area (VTA) and/or periaqueductal gray (PAG; Riters and Alger, 2004), which project to HVC (proper name), RA, and Area X (Appeltants et al., 2000, 2002; Castelino et al., 2007). There is also evidence that estrogens acting within HVC regulate song stereotypy (Meitzen et al., 2007). Aromatase (AROM) expression has been found within the POM in multiple songbirds, including canaries (Shen et al., 1995; Fusani et al., 2000), whereas AROM protein (in the absence of detectable AROM mRNA) has been found within presynaptic boutons within HVC in zebra finches (Peterson et al., 2005). Therefore, in addition to the potential actions of estrogens of a gonadal origin acting in the POM and HVC to regulate song, actions of estrogens generated from testosterone within these nuclei is also possible. There is also evidence that neural activity/hormone action in one region can influence morphological or neurophysiology characteristics in downstream or upstream brain regions (Brenowitz and Lent, 2002; Meitzen et al., 2007; Larson et al., 2013; Brenowitz, 2015; Alward et al., 2016c), which could affect control of song. All song features listed in italics are predicted to be controlled by androgens and/or estrogens based on the results of past studies (Spiro et al., 1999; Long and Fee, 2008; Picardo et al., 2016). HVC is colored as a gradient to represent the topographical nature of its functions: lateral HVC controls aspects of syllable usage, whereas medial HVC controls aspects of syntax (Basista et al., 2014). RA is also colored as gradient to represent the fact that it receives input from both HVC and LMAN, but then transforms these signals into firing patterns whose variability correlates to the variability of individual acoustic units of song (e.g., syllables; Spiro et al., 1999). LMAN is part of a circuit including Area X and the dorsolateral nucleus of the medial anterior thalamus (DLM) that is critical for song learning. RA projects to the tracheosyringeal portion of the twelfth cranial nerve (nXIIts), a hindbrain nucleus that contains androgen-sensitive motor neurons (Harding, 2008). nXIIts neurons control muscles of the syrinx, which are also androgen sensitive, to generate song. We have shown previously that androgens at the syrinx regulate aspects of vocal performance without affecting the motivation to sing or song stereotypy (Alward et al., 2016b). The basic principle of steroid hormone action in the regulation of a complex behavior presented here, that steroid hormones act in a nonredundant manner throughout the brain and periphery to regulate behavior, may be applicable to a wide-range of behaviors and systems. SCS, Song control system.
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