Manipulations of inhibition in cortical circuitry differentially affect spectral and temporal features of Bengalese finch song

Abstract

The interplay between inhibition and excitation can regulate behavioral expression and control, including the expression of communicative behaviors like birdsong. Computational models postulate varying degrees to which inhibition within vocal motor circuitry influences birdsong, but few studies have tested these models by manipulating inhibition. Here we enhanced and attenuated inhibition in the cortical nucleus HVC (used as proper name) of Bengalese finches (Lonchura striata var. domestica). Enhancement of inhibition (with muscimol) in HVC dose-dependently reduced the amount of song produced. Infusions of higher concentrations of muscimol caused some birds to produce spectrally degraded songs, whereas infusions of lower doses of muscimol led to the production of relatively normal (nondegraded) songs. However, the spectral and temporal structures of these nondegraded songs were significantly different from songs produced under control conditions. In particular, muscimol infusions decreased the frequency and amplitude of syllables, increased various measures of acoustic entropy, and increased the variability of syllable structure. Muscimol also increased sequence durations and the variability of syllable timing and syllable sequencing. Attenuation of inhibition (with bicuculline) in HVC led to changes to song distinct from and often opposite to enhancing inhibition. For example, in contrast to muscimol, bicuculline infusions increased syllable amplitude, frequency, and duration and decreased the variability of acoustic features. However, like muscimol, bicuculline increased the variability of syllable sequencing. These data highlight the importance of inhibition to the production of stereotyped vocalizations and demonstrate that changes to neural dynamics within cortical circuitry can differentially affect spectral and temporal features of song.NEW & NOTEWORTHY We reveal that manipulations of inhibition in the cortical nucleus HVC affect the structure, timing, and sequencing of syllables in Bengalese finch song. Enhancing and blocking inhibition led to opposite changes to the acoustic structure and timing of vocalizations, but both caused similar changes to vocal sequencing. These data provide support for computational models of song control but also motivate refinement of existing models to account for differential effects on syllable structure, timing, and sequencing.

Keywords: HVC; birdsong; sequencing; songbird; tempo.

Fig. 1.

Location of probes relative to HVC (n = 8 birds). The location of probes for different birds are plotted in different colors.

Fig. 2.

Muscimol reduced the amount of singing in a dose-dependent manner (n = 9 birds). The scatterplot depicts the number of songs produced during the 4-h experimental period following infusions of different doses of muscimol. Although raw values were analyzed, data are plotted as % of the average number of songs produced after phosphate-buffered saline (PBS) infusions to better visualize the effect of muscimol (song rate under control conditions varied across birds). Each bird is depicted with a different symbol. Song rate was significantly reduced after 1, 2, and 3 mM doses of muscimol (see results) but was not affected by lower doses (0.1–0.2 and 0.5 mM; mixed-effects model). *Indicates that song rate was significantly reduced for these concentrations during the 4-h experimental period. *P < 0.05 (mixed-effects model).

Fig. 3.

Higher doses of muscimol led to the production of “degraded” songs. A: examples of a spectrogram (top) and a raw sound trace (oscillogram) of songs (bottom) from a control [phosphate-buffered saline (PBS)] day (left) and from an adjacent day in which muscimol infusion led to the production of spectrally degraded song (right). Scale bars, 1 s. B: kernel density estimates [ggplot in R; bandwidth (c) of 0.9] depicting the data for 6 acoustic features after PBS or muscimol (MUSC) infusions for the bird depicted in A. C and D: changes to the median (C) and normalized interquartile region (normIQR; D) of acoustic features of degraded songs after muscimol infusions. Plotted are % changes or differences compared with normal songs following control infusions. Circles are scaled to values on left y-axis, and squares are scaled to values on right y-axis. The light gray symbols identify the data for example depicted in A. *P < 0.05 (mixed-effects model).

Fig. 4.

The median and variability of acoustic features of individual syllable labels within the nondegraded songs (n = 8 experiments across 8 birds) after muscimol infusions were different from those of control songs after phosphate-buffered saline (PBS) infusions. A: spectrograms of a song produced after PBS infusions (top) and a nondegraded song produced after muscimol infusions (bottom). White letters at top of spectrograms indicate labels assigned to syllables within the songs. Scale bar, 500 ms. B: scatterplots of % changes or differences (relative to control) in median values of acoustic features for each syllable type (“a,” “b,” ”c,” etc.; n = 94 syllables across 8 birds) after muscimol infusions. Muscimol infusions significantly decreased syllable amplitude and mean frequency and increased the spectral, spectrotemporal, and amplitude entropies of syllables. C: scatterplots of differences in the normalized interquartile region [normIQR (variability)] of acoustic features for each syllable type. Muscimol infusions increased the variability of all measured acoustic features except amplitude entropy. For both B and C, each circle corresponds to a syllable type in a bird’s song and was plotted with some jitter to facilitate visualization of data points. Raw data were analyzed, but because of the wide range in values across syllable labels, % changes and differences were plotted to facilitate data visualization. *P < 0.05 (mixed-effects model).

Fig. 5.

Muscimol increased the variability of syllable sequencing at branch points, and the magnitude of effect varied depending on the inherent variability of syllable sequencing of the branch point. A: the change in transition entropy caused by muscimol infusions for each branch point (n = 22 branch points across 8 birds) in relation to the transition entropy of the branch point under control conditions. There is a significant negative relationship between the degree to which muscimol infusions increased transition entropy and transition entropy under control conditions (mixed-effects model). Each symbol represents a different bird. B and C: examples of 2 branch points from a single bird, 1 with low transition entropy under control conditions (B) and another with high transition entropy under control conditions (C). For each example, on left are spectrograms of the transitions following the branch point sequence (“defgh” in B and “abc” in C), and on right are the transition probabilities (%) for each of the branch point transitions following phosphate-buffered saline (PBS) or muscimol infusions. Also listed are the transition entropies under PBS and muscimol conditions. The degree of change in transition entropy is greater for the branch point in B than the branch point in C.

Fig. 6.

Infusions of bicuculline methiodide (BMI; n = 5 birds) increased song rate at lower doses (1–3 mM) but tended to decrease song rate at higher doses (4–10 mM). The scatterplot depicts the number of songs produced during the 4-h experimental period following infusions of different doses of BMI. Although raw data were analyzed, data are plotted as % of the average number of songs produced after phosphate-buffered saline (PBS) infusions (see methods) to facilitate data visualization (mixed-effects model). Each bird is depicted with a different symbol. *P < 0.05 (mixed-effects model).

Fig. 7.

Infusions of bicuculline methiodide (BMI) affected the median and variability of acoustic features of individual syllable labels within song. A: spectrograms of a song produced after phosphate-buffered saline (PBS) infusions (top) and a nondegraded song produced after BMI infusions (bottom). White letters at top of spectrograms indicate labels assigned to syllables within the songs. Scale bar, 500 ms. B: scatterplots of % changes or differences (BMI vs. PBS) in median values of acoustic features for each syllable type (“a,” “b,” “c,” etc.; n = 66 syllables in 5 birds). BMI infusions significantly increased syllable duration, mean frequency, and amplitude and tended to increase spectral entropy. C: scatterplots of differences in the normalized interquartile region [normIQR (variability)] of acoustic features for each syllable type. BMI infusions decreased the variability of mean frequency and amplitude and tended to decrease the variability of spectrotemporal and amplitude entropy. For both B and C, each circle corresponds to a syllable label in a bird’s song and was plotted with some jitter to facilitate visualization of data points. Raw data were analyzed, but because of the wide range in values across syllable labels, % changes and differences were plotted to facilitate data visualization. *P < 0.05, ^~P < 0.10 (mixed-effects model); (*) indicates that the distribution was significantly different from zero when an outlying value was removed.

Fig. 8.

Bicuculline increased the variability of syllable sequencing at branch points, and the magnitude of effect varied depending on the inherent variability of syllable sequencing. Plotted is the change in transition entropy caused by bicuculline methiodide (BMI) infusions for each branch point (n = 15 branch points across 5 birds) in relation to the transition entropy of the branch point under control conditions. There was a significant negative relationship between the degree to which BMI infusions increased transition entropy and transition entropy under control conditions (mixed-effects model). Each symbol represents a different bird.