<i>Parvalbumin</i> (<i>PV</i>) specialization in dorsal arcopallial (dA) and nidopallial (dAN) nuclei in female downy woodpeckers, as well as 2 other woodpecker species.

(A, B) Female downy woodpecker. (C, D) Male red-bellied woodpecker. (E, F) Male hairy woodpecker. Shown are representative in situ hybridization microscope images (inverted black and white colormetric) of PV expression (white), with fast red as a counterstain (grey). Nido, nidopallium; Meso, mesopallium; Arco, arcopallium; Ot, optic tectum. Scale bars = 500 μm. White dashed lines in images indicate boundaries for different telencephalic regions. White dashed lines in high-magnification images indicate boundaries for different telencephalic regions (e.g., boundary between nidopallium and mesopallium), whereas blue dashed lines indicate specialized PV regions identified in woodpeckers. Photo credits: Female downy woodpecker from Ken Thomas (Public Domain via WikiMedia); male red-bellied woodpecker from Neal Lewis (Public Domain Mark 1.0 via Flickr); and male hairy woodpecker from David Whelan (Public Domain via WikiMedia).</p

Identification of forebrain structures with specialized <i>parvalbumin</i> (<i>PV</i>) mRNA expression.

(A-F) Representative radioactive in situ hybridization microscope images of PV mRNA in species representing 8 different avian orders (see S1 Fig for penguin and emu). PV-rich forebrain nuclei were present in only (E) vocal learning hummingbirds (positive control) and (F) downy woodpeckers. (Ei-iii) High magnification of 3 telencephalic “song control” nuclei in hummingbirds. (Fi) High magnification of the woodpecker drumming nucleus of the anterior nidopallium (dAN); and (Fii) drumming nucleus of arcopallium (dA). Each scale bar is equal to 2 mm. Neuroanatomical markers shown in “A” are as follows: Hyper, hyperpallium; Meso, mesopallium; Nido, nidopallium; GP, globus pallidus; T, Thalamus; Ot, optic tectum; St, striatum; Arco, arcopallium; Ento, entopallium. White dashed lines in high-magnification images (Ei-iii and Fi-ii) indicate boundaries for different telencephalic regions (e.g., boundary between nidopallium and mesopallium), whereas blue dashed lines indicate specialized PV regions identified in Anna’s hummingbirds and downy woodpeckers. Image credits: flamingo from Wilfredo Rodríguez; turaco from Edelmauswaldgeist; duck from Orso della campagna e Papera dello stagno; hawk from Cheva; hummingbird from Stickpen, and downy woodpecker from Greg Schechter. All image licenses: CC Public Domain via WikiMedia.</p

Parvalbumin cell count data.

(CSV)</p

Assessing striatal specializations and markers for the woodpecker arcopallium.

Two markers, (A) ETV1 and (B) Lhx9 (radioactive in situ hybridization), were used to delineate the boundary of the arcopallium and nidopallium. (C-F) Representative in situ hybridization images (inverted black and white colormetric) of (C and D) FoxP1 and (E and F) RGS12 in zebra finch and downy woodpecker. Both genes are significantly enriched in the zebra finch Area X (striatal nucleus); however, neither demarcates a specialized region within the woodpecker striatum. FoxP1 allows for the clear delineation of all nidopallial-striatal boundaries. Both reveal similar patterns to zebra finches (see [23]). Data from Lhx9 was collected through radioactive in situ hybridization (see Materials and methods for details). Hyp: hyperpallium; arco: arcopallium; nido: nidopallium; Meso: mesopallium. Scale bar, 500 μm. (TIF)</p

Activity induced <i>Arc</i> expression in the dorsal nucleus of the arcopallium (dA) of male downy woodpeckers positively correlates with drumming behavior.

(A-B) Representative low-magnification in situ hybridization microscope images of Arc mRNA expression (white), with fast red as a counterstain (grey) in downy woodpeckers that were either passively caught (A) or (B) after drumming during a simulated territorial encounter (STI). (C-F) Representative high-magnification examples of Arc mRNA image in (C) passively caught birds (n = 3) or birds that listened to STI playback (see Materials and methods) but either (D) did not produce vocalizations or drums (silent, n = 3), (E) produced only vocalizations and no drums (n = 2), or (F) produced drums (n = 10). White dashed lines in high-magnification images indicate boundaries between the arcopallium and nidopallium, whereas blue dashed lines indicate specialized PV regions identified in downy woodpeckers. PV-rich dA area on each section was determined from PV mRNA on an adjacent slide. (G) Violin plots (horizontal line denotes median) illustrating in Arc gene expression in the dA of downy woodpeckers caught after producing different behaviors. IEG expression significantly differed in the dA (F3,14 = 4.74, p = 0.02), with drumming birds having significantly greater Arc mRNA compared to all other groups (all relevant comparisons: p Arc mRNA expression in dA was positively correlated with the number of drums (p Arc expression and the total number of aggressive vocalizations (I) or flights (J). Significant correlations have solid lines (p S2 Data.</p

Activity induced <i>Arc</i> expression in the dorsal anterior nidopallium (dAN) of downy woodpeckers positively correlates with drumming behavior.

(A-B) Representative low-magnification in situ hybridization microscope images of Arc expression (white), with fast red as a counterstain (grey) in downy woodpeckers that were either passively caught (A) or (B) after drumming during a simulated territorial encounter (STI). (C-F) Representative high-magnification examples of Arc mRNA image in (C) passively caught birds (n = 3) or birds that listened to STI playback (see Materials and methods) but either (D) did not produce vocalizations or drums (silent, n = 3), (E) produced only vocalizations and no drums (n = 2), or (F) produced drums (n = 10). Dash blue outline represents the PV-rich dAN area on each section determined from PV mRNA on an adjacent section. (G) Violin plots (horizontal line denotes median) illustrating differences in Arc gene expression in the PV-rich dAN in male downy woodpeckers caught after producing different behaviors. Arc mRNA expression significantly differed in the dAN (F3,14 = 21.14, p Arc expression than all other groups (all relevant comparisons: p Arc mRNA expression in the dAN nucleus was positively correlated with the number of drums (p = 0.01). (I) No correlation was detected with the total number of aggressive vocalizations. Yet, (J) there was a marginally significant negative correlation with woodpeckers that flew less having greater Arc mRNA expression in dAN. Significant correlations denoted by solid lines (p PV regions identified in downy woodpeckers. Data for G-J can be found in S2 Data.</p

Coronal plane of the forebrain structures in male budgerigars (parrot) and downy woodpecker illustrating specialized <i>parvalbumin</i> (<i>PV</i>) mRNA expression, with such expression patterns absent in a Harris hawk.

(A) Representative radioactive in situ hybridization of microscope images of PV mRNA, in budgerigar (parrot) pallial song nuclei (NAO core, AAC core and shell, and NLC core and shell). Sections modified from Chakraborty and colleagues (2015) with permission from Dr. Jarvis, who is also an author on the current paper. (B) Coronal sections of the woodpecker brain showing the analogous locations for dAN in the anterior nidopallium and dNA in the arcopallium. (C) Comparable coronal sections in a Harris hawk that show PV expression in many positive control areas (see S1 Table). However, unlike the parrot and woodpecker, there was no specialized expression in the arco- or nidopallium. The in situs have cresyl violet as a counter stain. (TIFF)</p

Behavioral and normalized immediate early gene (ZENK and Arc) expression.

(CSV)</p

Primers used to generate downy woodpecker in situ probes.

(DOCX)</p

<i>Parvalbumin</i> (<i>PV</i>) specializations in the forebrain of male zebra finches and male downy woodpeckers.

(A) Representative microscope image from inverted black and white colormetric in situ hybridization at low magnification of zebra finch brain sections, showing specialized up-regulation of PV mRNA expression in 3 of the 7 telencephalic song control nuclei including RA, HVC, and LMAN. Schematic diagrams next to the microscope image illustrate key neural anatomical subdivisions. Dashed circle in the zebra finch schematic drawing is the relative location of Area X as determined by other molecular markers on adjacent sections (see S4 Fig). (B) High-magnification image of the zebra finch LMAN area. (C) Number of PV+ cells in 2 song control nuclei (RA and LMAN) compared to the expression in the surrounding arcopallium (intermediate arcopallium [AI]) or nidopallium (anterior nidopallium [AN]) in adult male zebra finches (n = 2). (D-I) Representative images from low (D, G) and high (E, H) magnification with schematic illustrations of neuroanatomical regions with PV (dA, DLN, and dAN). (F, I) Graphical illustrations of the significant up-regulation of PV in the male downy woodpecker in (F) dAN (n = 16; t30 = 9.06, p n = 16, t30 = 5.91, p PV regions identified in zebra finches and downy woodpeckers. Asterisk (*) denotes significant differences between putative drumming nucleus and the surrounding pallium (p S1 Data.</p