<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

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

<i>Parvalbumin</i> (<i>PV</i>) mRNA staining with radioactive in situ hybridization across species (Anna’s hummingbird, <i>Calypte anna</i>; Downy woodpecker, <i>Dryobates pubescens</i>; Harris hawk, <i>Parabuteo unicinctus</i>; American flamingo, <i>Phoenicopterus ruber</i>; Red-rested turaco, <i>Tauraco erythrolophus</i>; Domestic duck, <i>Anas platyrhynchos domesticus</i>; Emu, <i>Dromaius novaehollandiae</i>; Humbolt penguin, <i>Spheniscus humboldti</i>).

PV expression present in a brain area is denoted by a plus (+) sign; PV absent in a brain area is denoted by a minus (–) sign. (DOCX)</p

Movement and hearing-induced IEG expression in telencephalic regions of woodpeckers.

(A-B) Schematic diagrams of sagittal brain sections for (A) lateral and (B) medial brain nuclei. (C) Violin plots (horizontal line denotes group median) illustrating significant differences in Arc mRNA expression in 3 movement related brain regions (AI: F3,14 = 3.99, p = 0.03, AN: F3,14 = 3.83, p = 0.03, AM: F3,14 = 10.50, p Arc expression in the (D) AI, (F) AN, or (H) AM (all p-values > 0.05). No correlation was detected between the total number of measured behaviors (drums, whinny calls, and flights) produced during the STI and Arc expression in the (E) AI (p > 0.05), but significant positive correlations were detected in the (G) AN and (I) AM (both p-values p Arc mRNA in putative drumming nuclei (e.g., dA (t13 = 3.10, p 13 = 3.73, p Arc expression (white) with fast red as a counterstain (grey) in a downy woodpecker that was (L) passively caught or (m) a bird that drummed while listening to a drumming playback during the STI. (N) Violin plots (horizontal line denotes group median) illustrating significant differences in Arc mRNA expression in 2 auditory forebrain nuclei (CMM: F3,13 = 4.91, p = 0.02; NCM: F3,13 = 4.05, p = 0.03) of downy woodpeckers caught after hearing playbacks of drumming and either being silent in response or producing different behaviors. Data for C-K and N can be found in S2 Data.</p

Parvalbumin (PV) specialization in the male and female downy woodpecker arcopallium and nidopallium.

(A-D) Representative PV mRNA expression (green) from fluorescent in situ hybridization experiments at low-magnification (tile scan) and (Ai and Ci) high-magnification illustrations of neuroanatomical regions with PV up-regulation in the male downy woodpecker brain. (E and F) Representative PV mRNA staining in the DLN, dAN, and dA of a female downy woodpecker. Blue signal is a DAPI nuclear stain. All scale bars are 1 mm. Asterisks (*) indicate folds on tissue. Photo credits: male downy woodpecker from Greg Schechter, and female downy woodpecker from Ken Thomas (CC Public Domain via WikiMedia). (TIF)</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

<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

Parvalbumin (PV) specialization through the downy woodpecker arcopallium.

(A) Colormetric and (B) radioactive in situ hybridization illustrating specialized patterns of PV or (C) arcopallium-enriched Lim homeobox 9 (Lhx9) mRNA expression. Medial parasagittal sections through the downy woodpecker arcopallium show that PV has specialized expression in dA of the dorsal and intermediate arcopallium. These findings are consistent with the 2 different types of probe labeling and hybridization methods. Although Lhx9 demarcates most of the woodpecker arcopallium, it is largely absent in the anterior arcopallium, as seen in songbirds [18]. Dashed lines indicate the arcopallial boundary. Scale bars represent 500 μm. (TIFF)</p