Vocal experimentation in the juvenile songbird requires a basal ganglia circuit

Abstract

Songbirds learn their songs by trial-and-error experimentation, producing highly variable vocal output as juveniles. By comparing their own sounds to the song of a tutor, young songbirds gradually converge to a stable song that can be a remarkably good copy of the tutor song. Here we show that vocal variability in the learning songbird is induced by a basal-ganglia-related circuit, the output of which projects to the motor pathway via the lateral magnocellular nucleus of the nidopallium (LMAN). We found that pharmacological inactivation of LMAN dramatically reduced acoustic and sequence variability in the songs of juvenile zebra finches, doing so in a rapid and reversible manner. In addition, recordings from LMAN neurons projecting to the motor pathway revealed highly variable spiking activity across song renditions, showing that LMAN may act as a source of variability. Lastly, pharmacological blockade of synaptic inputs from LMAN to its target premotor area also reduced song variability. Our results establish that, in the juvenile songbird, the exploratory motor behavior required to learn a complex motor sequence is dependent on a dedicated neural circuit homologous to cortico-basal ganglia circuits in mammals.

Figure 1. Inactivation of LMAN Significantly Reduces Vocal Experimentation, Making the Otherwise Variable Song of the Juvenile Zebra Finch Highly Stereotyped

(A) Two major pathways in the vocal control system of the songbird. The motor pathway (gray) includes motor cortex analogs HVC and RA, while the AFP (white), a basal ganglia thalamo-cortical circuit, consists of Area X, the dorsolateral anterior thalamic nucleus (DLM), and LMAN, which, in turn, projects to RA. To inactivate the output of the AFP, injections of TTX and muscimol (red bolus) were made into LMAN. (B) Examples of a juvenile zebra finch song (57 dph) showing large variability in the sequence and acoustic structure of song syllables. (C) Inactivating LMAN with TTX produces an immediate reduction of sequence and acoustic variability, revealing a highly stereotyped song produced by the motor pathway. The song snippets shown in (B) and (C) are from consecutive song bouts, immediately before and 1 h after drug injection. Songs are displayed as spectral derivatives calculated as described [36]. The frequency range displayed is 0–8.6 kHz. For audio of song bouts before and during LMAN inactivation in this bird, refer to Audios S1 and S2, and S3 and S4, respectively.

Figure 2. Analysis of the Effect of Bilateral LMAN Inactivation on Song Variability

(A) Consecutive renditions of a repeating song motif of 0.5 s duration in a juvenile bird (59 dph) arranged vertically. Note the large variations in acoustic structure within individual syllables before LMAN inactivation (left). Following TTX injection into LMAN, the acoustic variability is dramatically reduced (middle), only to return to the original level by the following day (right). Numbers below each column indicate the variability index (See Materials and Methods section) calculated for the four renditions of the syllables shown. (B) Scatter plot of variability scores before and during LMAN inactivation with TTX (red) and muscimol (blue). Also shown are results for bilateral TTX injection into MMAN (black; see text), and saline injection into LMAN (green). (C) Time course of variability reduction following TTX (red) and muscimol (blue) injections show a time dependence that reflects the known in vivo pharmacology of the respective agents. Data were averaged over four identified syllables and taken from the same bird over consecutive days (dph = 70 and 71; muscimol inactivation followed by TTX inactivation). (D) Distribution of variability scores for all syllables analyzed in the TTX and muscimol experiments (25 unique syllables, six birds) before (black) and during (red) LMAN inactivation in juvenile birds. Shown for comparison are the variability scores for adult zebra finch syllables (18 syllables, 4 birds; undirected song, green; directed song, light blue). Dots represent raw data, while the lines are smoothed running averages. (E) TTX inactivation of LMAN significantly increased syllable sequence stereotypy. Sequence stereotypy scores (see Materials and Methods) for six birds before (black) and after (red) TTX injections into LMAN. For comparison, the average stereotypy score for adult birds singing directed song was 0.95 (n = 4 birds).

Figure 3. Song-Aligned Firing Patterns of RA-Projecting LMAN Neurons in Singing Juvenile Zebra Finches Are Highly Variable

(A) Three successive renditions of a 67-d-old bird's song motif. Displayed under each spectrogram is the simultaneously recorded voltage waveform of an antidromically identified RA-projecting LMAN neuron (verified by collision testing). Average syllable variability for the three motifs is 0.31. Motif alignment was done at the onset (yellow lines) of syllable C. (B) Raster plot showing the spike patterns for 50 consecutive motif renditions for the same cell as in (A). The motifs from (A) are indicated in green. (C) Relative frequency of inter-spike intervals during singing (black) and non-singing (blue) for all the 17 identified projection neurons (units are intervals per second; bin size is 0.04 log units). (D) Distribution of spike-train correlations across all pairs of motifs for the cell in (B) (solid red line). Correlations calculated with random time shifts added to the spike trains have a similar distribution (dashed red line; see Materials and Methods). Also shown is the correlation distribution for the population of identified projection neurons (solid black line; mean correlation indicated by solid arrowhead), and for the population with random time shifts added (dashed black line). In comparison, spike trains of neurons in premotor nucleus RA of the adult bird are highly stereotyped (from [23]; mean correlation indicated by open arrowhead).

Figure 4. Bilateral Injections of the NMDA Receptor Antagonist AP5 into RA Significantly Reduced Song Variability

(A) Excitatory synaptic inputs to RA from LMAN and HVC are mediated by a different mix of glutamate receptor types (see text). Using AP5 we could block LMAN input while only partially inactivating HVC input. (B) Eight sequential renditions of one song syllable in a juvenile zebra finch (63 dph) before and after AP5 injection into RA. Note the rapid fluctuations in pitch, the appearance of noisy acoustic structure, and variations in syllable duration before injection. The average variability scores (V) before and after injections for the eight shown syllable renditions were 0.50 and 0.25, respectively. (C) Following injection of AP5 into RA, fluctuations in acoustic structure were substantially reduced. Variability scores of 11 syllables in four birds before and after injection of AP5 into RA. (D) Time course of acoustic variability following drug injection averaged over all identifiable syllables for the bird in (B).