Convergent differential regulation of parvalbumin in the brains of vocal learners

Abstract

Spoken language and learned song are complex communication behaviors found in only a few species, including humans and three groups of distantly related birds--songbirds, parrots, and hummingbirds. Despite their large phylogenetic distances, these vocal learners show convergent behaviors and associated brain pathways for vocal communication. However, it is not clear whether this behavioral and anatomical convergence is associated with molecular convergence. Here we used oligo microarrays to screen for genes differentially regulated in brain nuclei necessary for producing learned vocalizations relative to adjacent brain areas that control other behaviors in avian vocal learners versus vocal non-learners. A top candidate gene in our screen was a calcium-binding protein, parvalbumin (PV). In situ hybridization verification revealed that PV was expressed significantly higher throughout the song motor pathway, including brainstem vocal motor neurons relative to the surrounding brain regions of all distantly related avian vocal learners. This differential expression was specific to PV and vocal learners, as it was not found in avian vocal non-learners nor for control genes in learners and non-learners. Similar to the vocal learning birds, higher PV up-regulation was found in the brainstem tongue motor neurons used for speech production in humans relative to a non-human primate, macaques. These results suggest repeated convergent evolution of differential PV up-regulation in the brains of vocal learners separated by more than 65-300 million years from a common ancestor and that the specialized behaviors of learned song and speech may require extra calcium buffering and signaling.

Figure 1. Avian phylogenic tree and schematic drawings of vocal learner and non-learner brains.

(A) Avian phylogenic tree. Shown are the branches for 27 major orders and one suborder (suboscines), highlighting 1–2 species each, based on the proposal of Hackett et al 2008 . Bold text, vocal learners. Black nodes, proposed independent gains of vocal learning. White node, an alternative possibility where there was two independent gains of vocal learning (hummingbirds and the common ancestor of parrots and songbirds), then lost in suboscine songbirds. (B) Schematic sagittal drawing of example vocal learner (songbird and human) and non-learner (quail and macaque) brains. Black lines, song motor pathway. White lines, pallial-basal-ganglia song pathway. Dashed lines, connections between the two pathways. Red line, direct projection from forebrain to brainstem vocal motor neurons found in vocal learners. Connections in humans are predicted based on known motor pathways in mammals, except the direct projection to Amb and nXII, which has been experimentally determined in humans. Non-human primates have what is called a pro-motor (ProM) region (or laryngeal motor cortex) in the premotor cortex that makes an indirect projection to Amb, but unlike vocal learners this region is not required nor appears to influence vocalizations. For reviews, see Jurgens (2002) , Jarvis (2004) , Fitch et al (2010) , and Simonyan et al (2011) . Abbreviations: Am or Amb, nucleus ambiguus Area X, a vocal nucleus (no abbreviation) ASt, anterior striatum AT, anterior thalamus DLM, dorsal lateral nucleus of the thalamus DM, dorsal medial nucleus of the midbrain FMC, face motor cortex H, hindbrain HVC, a vocal nucleus (no abbreviation) LMAN, lateral magnocellular nucleus of the anterior nidopallium M, midbrain, nXII, 12^th motor nucleus PAG, periaqueductal gray PFC, prefrontal cortex ProM, promoter laryngeal cortex in non-human primates RA, robust nucleus of the arcopallium RF, reticular formation T, thalamus V, ventricle.

Figure 2. Microarray results for PV expression.

(A) Seven different PV oligos (symbols) in the top 100 candidate gene transcripts were differentially expressed between vocal learners and non-learners in the arcopallium. (B) Example PV oligo that showed lower PV expression in nXllts compared to SSp in hummingbird, dove, and quail. Y-axes, log2 fold expression ratio of the arcopallium song nucleus or ciA versus the adjacent iA, or SSp relative to nXIIts; ratio = 0 means no difference between brain areas. Clone IDs of cDNAs that the oligos recognize are in http://songbirdtranscriptome.net/and http://aviangenomes.org. ZF, zebra finch; BG, budgerigar; H, hummingbird; D, dove; Q, quail. **p<0.01 paired t-test on each oligo of each group of species that share a trait (n = 9 vocal learners; n = 5 vocal non-learners; empirical Bayes adjusted paired t-test; the data shown is for averages of each species). **p<0.01, unpaired t-test, combined values of PV oligo variants.

Figure 3. PV mRNA expression in vocal motor pathway nuclei in avian species.

(A1–5) Darkfield images of PV expression in the arcopallium (delineated by dashed white lines) of three vocal learners and two non-learners. The arcopallium song nuclei are highlighted (RA, AAc, and VA) as well as the iA region lateral or medial used to compare with in the microarray and in-situ hybridization experiments. The nidopallium song motor nucleus (HVC, NLC, and VLN) of each species is also highlighted. (B1–10) Brightfield images of the arcopallium song nucleus for vocal learners and central intermediate arcopallium (ciA) in non-learners (odd numbers), and the arcopallium area adjacent to the song nucleus or ciA (even numbers). (C1–5) Darkfield images of PV expression in nXIIts and SSp for each species. (D1–10) Brightfield images of SSp (odd numbers) and nXllts (even numbers) for each species. Scale bar in D10 = 30 µm (applies to all brightfield images).

Figure 4. PV mRNA expression ratios measured from the in situ hybridizations in avian species.

(A) PV expression ratio of arcopallium song nuclei and adjacent intermediate arcopallium (iA) for vocal learners, and ciA and adjacent intermediate arcopallium (iA) for non-learners. Also quantified is a control gene, ER81. The line across the graph is at ratio = 1, which means both areas equally express the gene. (B) PV expression ratio of SSp and nXllts, and another control gene GDNF family receptor alpha 1. Stars (*) inside bars indicate significant difference between two areas (A. arcopallium song nuclei or ciA and surrounding iA. B. SSp and nXllts) within each species using paired t-test on raw values. Number symbol (#) above bars indicates a significant difference between vocal learners and non-learners using unpaired t-test on ratios. ZF, zebra finch; BG, budgerigar; H, hummingbird; Hl, humming bird lateral part of nXllts; Hm, hummingbird medial part of nXllts; D, dove; Q, quail. *p<0.05, **p<0.001.

Figure 5. mRNA expression of control genes in avian vocal learners and non-learners.

(A–E) ER81, used as an arcopallium control gene. (F–J) GDNF family receptor alpha 1, used as a brainstem motor neuron control gene. Species are labeled above the panels. Scale bar = 2 mm (applies to all the images).

Figure 6. PV mRNA expression in pallial-basal ganglia song pathway nuclei of avian species.

(A) PV expression in anterior pathway song nuclei (LMAN and Area X) of zebra finch. (B) PV expression in anterior pathway song nuclei (NAo and MMSt) of budgerigar. (C) PV expression in anterior pathway song nuclei (VAN and VASt) in Anna's hummingbird. Higher PV expression was found in the anterior pathway pallial song nuclei of two species only, zebra finch and Anna's hummingbird. Images are representative of three animals each species. Scale bar = 2 mm (applies to all the images).

Figure 7. PV and GDNF family receptor alpha 1 mRNA expression in human and macaque brainstem.

(A–C) PV expression in human, including human nXll, Amb, and sensory nuclei; (D–F) GDNF family receptor alpha 1 expression in human identifying nXII and Amb motor neuron populations. (G–I) PV expression in macaque showing the homologous brain regions; (J–L) GDNF family receptor alpha 1 expression in macaque to identify the motor neuron populations. The first column (A, D, G, J) shows low magnification of the entire brainstem section, the second column (B, E, H, K) shows the location of nXII and the third (C, F, I, L) shows the location of Amb. Note the high PV expression in human nXII and low in macaque nXII, but more comparable expression of GDNF family receptor alpha 1 in both species. Scale bar = 2 mm (applied to D, E, F, J) and 1 mm (applied to K, L).

Figure 8. High power view and quantification of PV in nXII motor neurons of human and macaque.

(A–F) Darkfield view (inverted from brightfield) of labeled (white arrows; silver-white grains over cell bodies) and non-labeled (red arrow) cells in nXII, sensory nuclei (Gr), and adjacent reticular (RT) formation of human (A–C) and macaque (D–F). Images are from the same human (A–C) and macaque (D–F) individuals for accurate comparison, but are representative of all individuals. Many cells with high PV expression were observed in human nXII and sensory nuclei, but in macaque only few were seen in nXII, mainly at the periphery of the nucleus. (G) Percent of XII motor neurons with PV expression levels at ∼25% of or greater than seen in the sensory neurons of the same individual human or macaque. Motor neurons were recognized by their large size. (H) Ratio of the PV expression (% area of silver grains over cell bodies) of the XII motor neurons divided by the average of the sensory neurons for each individual human and macaque. * p-values are from unpaired t-test. Scale bar = 30 µm (applies to all the images).