Singing modulates parvalbumin interneurons throughout songbird forebrain vocal control circuitry

<div><p>Across species, the performance of vocal signals can be modulated by the social environment. Zebra finches, for example, adjust their song performance when singing to females (‘female-directed’ or FD song) compared to when singing in isolation (‘undirected’ or UD song). These changes are salient, as females prefer the FD song over the UD song. Despite the importance of these performance changes, the neural mechanisms underlying this social modulation remain poorly understood. Previous work in finches has established that expression of the immediate early gene EGR1 is increased during singing and modulated by social context within the vocal control circuitry. Here, we examined whether particular neural subpopulations within those vocal control regions exhibit similar modulations of EGR1 expression. We compared EGR1 expression in neurons expressing parvalbumin (PV), a calcium buffer that modulates network plasticity and homeostasis, among males that performed FD song, males that produced UD song, or males that did not sing. We found that, overall, singing but not social context significantly affected EGR1 expression in PV neurons throughout the vocal control nuclei. We observed differences in EGR1 expression between two classes of PV interneurons in the basal ganglia nucleus Area X. Additionally, we found that singing altered the amount of PV expression in neurons in HVC and Area X and that distinct PV interneuron types in Area X exhibited different patterns of modulation by singing. These data indicate that throughout the vocal control circuitry the singing-related regulation of EGR1 expression in PV neurons may be less influenced by social context than in other neuron types and raise the possibility of cell-type specific differences in plasticity and calcium buffering.</p></div

Diagram of the connections in songbird vocal control circuitry.

<p>Illustrated are HVC (proper name) and the robust nucleus of the arcopallium (RA) in the vocal motor pathway (white circles) and the basal ganglia nucleus Area X, the dorsolateral anterior thalamic nucleus (DLM), and the cortical nucleus, the lateral magnocellular nucleus of the anterior nidopallium (LMAN) in the anterior forebrain pathway (gray circles). (B) Photomicrograph of PV neurons (green, 488 filter) and EGR1 neurons (red; 596 filter) in Area X during undirected singing. Yellow arrows indicate colocalization. White scale bar = 25 μm.</p

Parvalbumin (PV) luminance is modulated by singing in HVC and Area X.

<p>PV luminance in HVC-lat and Area X differed between non-singing birds (NS; gray bars) and singing birds (female-directed singers, FD; dark green bars; undirected singers, UD; light green bars). Bars represent means, while circles correspond to individual data points. * indicates a significant difference at p<0.05, # indicates a trend toward a difference at p<0.10.</p

Percent of PV neurons expressing EGR1 across nuclei is modulated by singing.

<p>Percent of PV neurons expressing EGR1 in non-singing birds (NS; gray bars), birds producing female-directed song (FD; dark green bars) and birds producing undirected song (UD; light green bars). Box-and-whisker plots for each experimental group. Each box spans the interquartile range, horizontal black lines indicate the median and whiskers show the minima and maxima. Lines above bars indicate significance of post-hoc contrasts for brain areas in which experimental groups significantly differed. * indicates a significant difference at p<0.05; # indicates a trend toward a difference at p<0.10.</p

Modulation of EGR1 protein by singing and social context varies across the song system.

<p>EGR1 expression among non-singing birds (NS; gray bars), birds producing female-directed song (FD; dark green bars) and birds producing undirected song (UD; light green bars). Box-and-whisker plots for each experimental group. Each box spans the interquartile range, horizontal black lines indicate the median and whiskers show the minima and maxima. Lines above bars indicate significance of post-hoc contrasts for brain areas in which experimental groups significantly differed. * indicates a significant difference at p<0.05; # indicates a trend toward a difference at p<0.10.</p