Activity in a cortical-basal ganglia circuit for song is required for social context-dependent vocal variability

Abstract

Variability in adult motor output is important for enabling animals to respond to changing external conditions. Songbirds are useful for studying variability because they alter the amount of variation in their song depending on social context. When an adult zebra finch male sings to a female ("directed"), his song is highly stereotyped, but when he sings alone ("undirected"), his song varies across renditions. Lesions of the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the output nucleus of a cortical-basal ganglia circuit for song, reduce song variability to that of the stereotyped "performance" state. However, such lesions not only eliminate LMAN's synaptic input to its targets, but can also cause structural or physiological changes in connected brain regions, and thus cannot assess whether the acute activity of LMAN is important for social modulation of adult song variability. To evaluate the effects of ongoing LMAN activity, we reversibly silenced LMAN in singing zebra finches by bilateral reverse microdialysis of the GABA(A) receptor agonist muscimol. We found that LMAN inactivation acutely reduced undirected song variability, both across and even within syllable renditions, to the level of directed song variability in all birds examined. Song variability returned to pre-muscimol inactivation levels after drug washout. However, unlike LMAN lesions, LMAN inactivation did not eliminate social context effects on song tempo in adult birds. These results indicate that the activity of LMAN neurons acutely and actively generates social context-dependent increases in adult song variability but that social regulation of tempo is more complex.

Fig. 1.

A: diagram of the major nuclei of the song system. The motor pathway is shown in gray, the anterior forebrain pathway (AFP) in black. In the motor pathway, neurons of the cortex-like nucleus HVC (abbreviation used as proper name) synapse on neurons of the robust nucleus of the arcopallium (RA), which directly and indirectly project to motor neurons that innervate the syrinx and respiratory muscles. In the AFP, HVC provides input into the striato-pallidal nucleus Area X, which projects to the medial nucleus of the dorsolateral thalamus (DLM). DLM sends excitatory projections to the lateral magnocellular nucleus of the anterior nidopallium (LMAN). LMAN projects both back to Area X, and on to RA. B: camera lucida tracing of 1 coronal section showing spread of biotinylated muscimol (gray shading). LMAN and Area X were delineated by Nissl stain (see methods).

Fig. 2.

A, left: a representative spectrogram (frequency vs. time) of a song syllable sung many times during 1 experiment. Black dashed lines indicate the short portion of this syllable used for FF measurement. Right: a plot showing SDs of fundamental frequency (FF) of this syllable measured in both social contexts, before (“pre”), during (“muscimol”) and after (“post”) LMAN inactivation. Error bars indicate 95% CI of the SD. Variability of FF was much higher during directed song than undirected song in the pre-muscimol condition (P < 0.0001). During LMAN inactivation, the SD of FF during undirected song decreased dramatically, so that it equaled that of directed song (P = 0.5723). Post-muscimol, the difference in variability of FF returned (P < 0.0001). B: syllable from a different bird with a different FF and a longer duration constant-frequency portion. In the pre- and post-muscimol treatment conditions, the SD of FF was significantly higher during undirected song (P = 0.0023 and P = 0.0029, respectively). During LMAN inactivation, SD of FF was similar in both contexts (P = 0.0933). All preceding statistics are F test for equality of variance. ***P < 0.0001, **P < 0.005. Mean FF measurements (not shown) for syllable A: pre_undir = 637.9; pre_dir = 634.3; muscimol_undir = 631.1; muscimol_dir = 631.6; post_undir = 630.8; post_dir = 631.9. For syllable B: pre_undir = 1,698.2; pre_dir = 1,692.9; muscimol_undir = 1,690.3; muscimol_dir = 1,684.3; post_undir = 1,695.2; post_dir = 1,691.8. ANOVA followed by post hoc paired t-test did not find significant social modulation of mean FF for either syllable.

Fig. 3.

LMAN inactivation reversibly eliminates social context-dependent variability of syllable structure. Plots of variability of FF during undirected song vs. variability of FF during directed song. Each symbol represents 1 syllable recorded in both social contexts during 1 treatment condition. In 2 birds, we repeated the experiment several days after the 1st muscimol treatment. The syllables recorded in the 2nd experiment are indicated by open circles. The diagonal line represents equal variability in both social contexts. A: blue symbols represent the coefficient of variation (CV) of FF for 29 syllables recorded prior to drug. Variability was higher in the undirected context. In contrast, variability was similar in both social contexts during LMAN inactivation (red symbols: CV of FF for the same 29 syllables recorded in the muscimol condition). B: comparison of variability before muscimol to variability after muscimol washout. Green symbols representing CV of FF for the same 29 syllables post-muscimol indicate that variability of song structure was again higher in the undirected social context than in the directed.

Fig. 4.

A: variability of syllable structure measured by experiment. The CV of FF for all syllables measured in each individual bird, in each condition, were averaged to give a “per bird” score (squares; dotted lines connect undirected and directed data points for each bird). Bars indicate the mean per bird score for each condition (n = 5 birds; error bars indicate ± SE). Variability of syllable structure was higher during undirected song than directed song both pre- and post-muscimol (pre: P < 0.0001; post: P = 0.0010; post hoc paired t-test). Social modulation of the variability of syllable structure was eliminated by LMAN inactivation (P = 0.4620). ***P < 0.0001, **P < 0.005. B: black dots represent the FF of 1 syllable measured during undirected song over several hours. Between 10:30 and 15:30, FF ranged over 50 Hz. However, after muscimol entered the microdialysis probes (∼15:20), the range of FF narrowed within 30 min and remained tightly distributed throughout muscimol dialysis (indicated by arrow). Diamonds indicate the CV of FF calculated at 20 min intervals. During the 2nd 20 min interval after LMAN inactivation, the CV was lower than during any previous interval that day. When data were combined across the whole treatment period, the overall CV pre-muscimol was 0.019 (n = 183), while during muscimol it was 0.011 (P < 0.0001). For comparison, the CV of directed song in this bird pre-muscimol was 0.011. The break in the x axis indicates a period during which the bird did not sing undirected song.

Fig. 5.

LMAN inactivation reversibly eliminates social context-dependent spectral entropy. A and B: plots of mean spectral entropy during undirected song vs. mean spectral entropy during directed song. Each symbol represents 1 syllable recorded in both social contexts during 1 treatment condition. In 2 birds, we repeated the experiment several days after the 1st muscimol treatment. The syllables recorded in the 2nd experiment are indicated by open circles. The diagonal line represents equal spectral entropy in both social contexts. A: blue symbols represent mean spectral entropy for 29 syllables recorded prior to muscimol. Spectral entropy was higher in the undirected context. In contrast, entropy was similar in both social contexts during LMAN inactivation (red symbols: mean spectral entropy for the same 29 syllables, recorded during muscimol). B: comparison of spectral entropy before muscimol to spectral entropy after muscimol washout. Green symbols representing spectral entropy for the same 29 syllables post-muscimol indicate that entropy of syllable structure was again higher in the undirected social context than in the directed. C: plot of mean spectral entropy during muscimol vs. mean spectral entropy pre-muscimol. During directed song, syllables have approximately the same spectral entropy with and without drug, as shown by the even distribution of purple symbols along the diagonal (P = 0.1689, Wilcoxon signed-rank test, n = 29). However, during undirected song, spectral entropy is lower in syllables produced during LMAN inactivation (gray symbols), and the ratio of muscimol: pre-muscimol spectral entropy is significantly <1 (P = 0.0010).

Fig. 6.

LMAN inactivation did not affect the social modulation of several supra-syllabic features of song. A: number of introductory notes per bout. Solid squares indicate the number of introductory notes per bout during undirected song for 1 bird, and open squares indicate the number during directed song. For A–D, dotted lines connect the data points for each bird, and bars indicate the mean group number for each condition (±SE). The number of introductory notes produced per bout was always higher during directed song even during muscimol treatment. B: CV of introductory notes per bout. In 3 of 4 birds, there was no difference in CV between directed and undirected song, and across birds there was no effect of LMAN inactivation on the variability in the number of introductory notes produced per bout of song (P = 0.7327, repeated measures 2-way ANOVA). C: number of motifs per bout. Solid squares indicate the number of motifs per bout during undirected song for 1 bird, and open squares indicate the number during directed song. Four of 5 birds sang more motifs per bout during directed song than during undirected song. One bird sang more motifs per bout in the undirected context. Neither pattern changed during LMAN inactivation. D: CV of the number of motifs per bout. The number of motifs produced per bout of song was more variable during directed song, and this remained true during muscimol treatment. **P < 0.01, *P < 0.05.

Fig. 7.

LMAN inactivation did not affect song tempo. ●, the ratio of mean undirected motif duration to the mean directed motif duration for 1 bird; …, connecting data points for the same bird during different treatments. A data point above 1 indicates that undirected song tempo is slower than directed song tempo. Bars indicate group means; error bars indicate SE. There are no differences in the means for each treatment (n = 5 birds; P = 0.2297, repeated measures ANOVA).