Summary of primer test and efficiency for each primer pair and species.

<p>Summary of primer test and efficiency for each primer pair and species.</p

Supplementary material for Heim et al.: 1. Auditory discrimination learning and acoustic cue weighing in female zebra finches with localised FoxP1 knockdowns

Rare disruptions of the transcription factor FoxP1 are implicated in a human neurodevelopmental disorder characterised by autism and/or intellectual disability with prominent problems in speech and language abilities. Avian orthologues of this transcription factor are evolutionarily conserved and highly expressed in specific regions of songbird brains, including areas associated with vocal production learning and auditory perception. Here, we investigated possible contributions of FoxP1 to song discrimination and auditory perception in juvenile and adult female zebra finches. They received lentiviral knockdowns of FoxP1 in one of two brain areas involved in auditory stimulus processing, HVC (proper name) or CMM (caudomedial mesopallium). Ninety-six females, distributed over different experimental and control groups were trained to discriminate between two stimulus songs in an operant Go/Nogo paradigm and subsequently tested with an array of stimuli. This made it possible to assess how well they recognised and categorised altered versions of training stimuli and whether localised FoxP1 knockdowns affected the role of different features during discrimination and categorisation of song. Although FoxP1 expression was significantly reduced by the knockdowns, neither discrimination of the stimulus songs nor categorisation of songs modified in pitch, sequential order of syllables or by reversed playback were affected. Subsequently, we analysed the full dataset to assess the impact of the different stimulus manipulations for cue weighing in song discrimination. Our findings show that zebra finches rely on multiple parameters for song discrimination, but with relatively more prominent roles for spectral parameters and syllable sequencing as cues for song discrimination.</p

Stability ranking of expression analysis for the song data set with three different statistical methods.

<p>Stability ranking of expression analysis for the song data set with three different statistical methods.</p

Expression levels of candidate reference genes for the zebra finch song (A–C) and tissue dataset (D–H).

<p>Values are given as cycle threshold numbers (C_t values) in (A) all Area X samples combined (n = 12), (B) silent group only (n = 7), (C) singing group only (n = 5), (D) tissue dataset all samples (n = 32), (E) male brain only (n = 10), (F) female brain only (n = 10), (G) testes only (n = 6) and (H) ovaries only (n = 6).</p

Specificity of primers for <i>SDHA</i> in qRT-PCR of nine avian species.

<p>Melting curves of five dilutions run in triplicates are shown with one panel per species. Species names are indicated in the title of each panel.</p

Specificity of 10 reference genes of the zebra finch with single peaks in melting curves.

<p>All amplifications were run in triplicate on 32 tissue samples.</p

Results of testing the stability of expression analysis for the four different zebra finch tissues and three different statistical methods.

<p>Results of testing the stability of expression analysis for the four different zebra finch tissues and three different statistical methods.</p

Sequence similarity of all amplified products to the chicken (ICGSC Gallus gallus 4.0) and zebra finch (WUGSC 3.2.4/taeGut1) genomes.

<p>Sequence similarity of all amplified products to the chicken (ICGSC Gallus gallus 4.0) and zebra finch (WUGSC 3.2.4/taeGut1) genomes.</p

Abbreviations for zebra finch brain structures.

<p>Abbreviations for zebra finch brain structures.</p

Audio_Bird5

Audio_Bird