The cultural transmission of behaviour depends on the ability of the pupil to identify and emulate an appropriate tutor1,2,3,4. How the brain of the pupil detects a suitable tutor and encodes the behaviour of the tutor is largely unknown. Juvenile zebra finches readily copy the songs of the adult tutors that they interact with, but not the songs that they listen to passively through a speaker5,6, indicating that social cues generated by the tutor facilitate song imitation. Here we show that neurons in the midbrain periaqueductal grey of juvenile finches are selectively excited by a singing tutor and—by releasing dopamine in the cortical song nucleus HVC—help to encode the song representations of the tutor used for vocal copying. Blocking dopamine signalling in the HVC of the pupil during tutoring blocked copying, whereas pairing stimulation of periaqueductal grey terminals in the HVC with a song played through a speaker was sufficient to drive copying. Exposure to a singing tutor triggered the rapid emergence of responses to the tutor song in the HVC of the pupil and a rapid increase in the complexity of the song of the pupil, an early signature of song copying7,8. These findings reveal that a dopaminergic mesocortical circuit detects the presence of a tutor and helps to encode the performance of the tutor, facilitating the cultural transmission of vocal behaviour.
The cortical song nucleus HVC is crucial for singing and song learning7,9,10,11,12 and receives convergent input from premotor, auditory and neuromodulatory neurons, including dopamine (DA)-secreting neurons in the midbrain periaqueductal grey (PAG)13,14,15 (Fig. 1a–c and Extended Data Fig. 1a–c). In the mammalian PAG, DA neurons encode information about social context, arousal in response to behaviourally salient stimuli, or rewards16,17,18, raising the possibility that the PAG-to-HVC pathway in juvenile finches encodes information about the tutor that facilitates song imitation. To explore this idea, we implanted tetrodes into the PAG of juvenile male finches raised in isolation from a tutor (tutor-naive juveniles; see Methods) (Fig. 1d–k). Most PAG neurons (81.8%; 18 out of 22 neurons from four birds) increased their action potential activity in the presence of a singing tutor (Fig. 1e–g, k), whereas PAG activity was unaffected during encounters with non-singing adult male finches or female finches, which do not sing (Fig. 1i–k). Neural activity in the PAG of the juvenile was not precisely locked to syllables of the song of the tutor, was variable across different tutor song bouts, and could remain elevated for hundreds of milliseconds after the tutor had stopped singing (Extended Data Fig. 2c–f), suggesting that PAG activity evoked by a singing tutor is not simply auditory in nature. Indeed, playback of the song of an adult finch from a speaker, including that of a recent tutor, failed to evoke activity in the PAG of the juvenile (Fig. 1h, k). Moreover, song playback from a speaker in the presence of an adult female bird failed to activate PAG neurons in tutor-naive juveniles (Extended Data Fig. 2a, b). Therefore, PAG neurons in juvenile males respond strongly and selectively to a live singing tutor and can thus signal the presence of a suitable song model.
These findings raise the possibility that experience of a singing tutor stimulates DA release from PAG terminals in the HVC. We explored this idea by virally expressing a modified dopamine type 2 (D2) receptor in HVC neurons of tutor-naive juvenile males that increases fluorescence upon DA binding (Fig. 2) (AAV2/9-GRABDA1h)19. We then head-fixed these juvenile males in the awake state and used two-photon imaging methods20 to establish that DA levels in the HVC increase in the presence of a singing tutor (Fig. 2c–d, i). By contrast, DA-related changes in fluorescence were not detected in the HVC of the juvenile in response to song playback (Fig. 2e, i), or when the juvenile encountered non-singing adult males or females (Fig. 2f, g, i), paralleling the selective enhancement of PAG activity elicited by a singing tutor. Moreover, ablating DA neurons in the PAG of the pupil with 6-hydroxydopamine (6-OHDA21) prevented tutor-evoked DA transients in the HVC of the pupil (Fig. 2h, i), confirming that tutor-evoked DA release in the HVC of the pupil largely originates from the PAG.
To explore whether DA signalling in the HVC has a role in song imitation, we used 6-OHDA to lesion DA-releasing fibres in the HVC of juvenile male finches raised continuously with adult male tutors and tracked their song development into adulthood (Fig. 3a–c and Extended Data Fig. 3). Lesions of DA-releasing fibres in HVC made near the onset of the sensitive period for tutor song memorization (30 days after hatching22) prevented song copying (Fig. 3d, e) without affecting the overall rate of singing (Extended Data Fig. 4a). As adults, these 6-OHDA-treated birds produced abnormally long and acoustically simple syllables, similar to finches raised in isolation from a tutor22 (Extended Data Fig. 4b, c). The 6-OHDA lesions made in the HVC in 30-day-old males are permanent and thus could potentially interfere with tutor song memorization (that is, sensory learning), the subsequent phase of song copying (sensorimotor learning), or both. However, 6-OHDA lesions made in the HVC of 45-day-old males, which have had sufficient tutoring experience to enable accurate copying but are just beginning sensorimotor learning22, did not affect the ability of a juvenile to copy the song of a tutor (Fig. 3d, f).
These findings suggest that DA signalling in the HVC has a role in sensory learning, but we cannot exclude a more general but developmentally restricted (before 45 days of age, for example) role for such signalling. Therefore, we used microdialysis methods23 to reversibly block DA receptors in the HVC24 of tutor-naive juvenile males (age: 43.0 ± 4.9 days of age (mean ± s.d.), n = 5) while they were housed with a tutor for 1.5 h on five consecutive days, allowing us to better determine whether DA signalling in the HVC is crucial during pupil–tutor interactions, when sensory learning occurs (Fig. 3g, h and Extended Data Fig. 5a–c). Reversibly blocking DA receptors in the HVC during, but not immediately after, tutoring sessions blocked song copying (Fig. 3h and Extended Data Fig. 5b, c), without affecting attentive behaviours of juveniles to tutors or the singing rates of tutors (Extended Data Fig. 5d, e and Supplementary Videos 1, 2). Moreover, reversibly suppressing PAG activity in the pupil with muscimol during daily tutoring sessions also blocked song copying; notably, juveniles in which the PAG was inactivated also failed to orient to their tutors, even though tutors continued singing at normal rates (Extended Data Fig. 5d–h and Supplementary Video 3). Thus, tutor-evoked activation of the PAG of the pupil and concomitant release of DA in the HVC are essential to encoding tutor song experience, and PAG activity may be required for the pupil to attend to a singing tutor.
The current findings do not exclude the possibility that DA signalling at other sites also contributes to sensory learning. One potential site is the basal ganglia region, Area X11, which receives dopaminergic input from the ventral tegmental area and substantia nigra pars compacta, as well as from a smaller cohort of TH+ PAG neurons (Extended Data Fig. 1d–g), and where dopamine signalling has a role in sensorimotor learning25. Nevertheless, infusing DA receptor blockers into Area X of juvenile males during daily tutoring sessions did not affect song copying (Extended Data Fig. 6). Another potential site is the caudal mesopallium, an auditory forebrain region important for song memory26,27. However, blocking DA receptors in the caudal mesopallium of juvenile males during daily tutoring sessions did not block song copying (Extended Data Fig. 5i–k).
These results show that DA release from PAG axon terminals in the HVC (PAGHVC terminals) signals the presence of a suitable model and helps to encode this model in the brain of the pupil. Consequently, artificially activating PAGHVC terminals should compensate for the absence of a live tutor and facilitate vocal copying in response to song playback. To test this hypothesis, we used adeno-associated viruses (AAVs) to express channelrhodopsin-2 (ChR2) bilaterally in the PAG of tutor-naive juvenile males (Fig. 3i, j and Extended Data Fig. 7a–d). Several weeks (33.3 ± 7.4 days (mean ± s.d.), n = 6) later, we implanted optical fibres bilaterally over the HVC and optogenetically activated PAGHVC terminals while playing the song of an adult male zebra finch through a speaker. Pairing PAGHVC terminal stimulation with song playback resulted in a significant level of song copying compared to juveniles that had only been exposed to song playback, or to song playback and optical illumination of HVC in the absence of ChR2 (Fig. 3j and Extended Data Fig. 7b; see Methods). Moreover, the combination of song playback and PAGHVC terminal stimulation while infusing DA blockers into the HVC did not lead to song copying in tutor-naive juveniles (Extended Data Fig. 7e–g).
To explore how tutor-evoked DA release from PAGHVC axon terminals alters HVC to drive song imitation, we implanted tetrodes in the HVC of tutor-naive juveniles and recorded neural activity before and after their initial encounters with a singing tutor (Fig. 4a–f). Spontaneous burst firing in HVC neurons increased within 1 h after the initial exposure of the juvenile to a singing tutor (Fig. 4b, c, e), without any change in their mean firing rates (Extended Data Fig. 8d). Because burst firing in HVC is driven by auditory afferent neurons12, this enhanced burst firing suggests that tutoring rapidly potentiates auditory inputs to the HVC. In fact, brief (35.0 ± 16.8 min (mean ± s.d.)) experience with a singing tutor led rapidly (around 1 h) to the emergence of temporally precise responses in the HVC of an awake juvenile to tutor song playback (Fig. 4d, f and Extended Data Fig. 8a–c). Furthermore, the mean firing rate of HVC neurons to song playback was unaffected by tutoring (Extended Data Fig. 8e, f), indicating that neural responses in the HVC became more tightly locked to specific features in the tutor song. None of these juveniles (n = 4) sang during or for several hours after the tutoring session, and thus these physiological changes were not simply the result of auditory feedback associated with vocal rehearsal. In another set of tutor-naive juvenile males, we found that tutoring rapidly reduced the kurtosis of vocal duration (Fig. 4g, h) and increased the mean entropy variance of the songs of the juveniles (Fig. 4i), two early hallmarks of song copying7,8. Notably, blocking DA signalling in the HVC of the pupil with 6-OHDA or DA blockers prevented these physiological and behavioural changes (Fig. 4e, f, h, i).
The discovery that DA neurons in the PAG of the pupil are strongly and selectively activated by a singing tutor parallels emerging evidence that potentially homologous neurons in the mammal can encode social cues, including those related to reward, context or novelty16,17. Indeed, the present findings advance a model in which both social cues and the song-related auditory input provided by the singing tutor drive the coincident activation of DA receptors and auditory synapses in the HVC, leading to the rapid emergence of auditory representations of the song of the tutor necessary to song imitation10,20 (Extended Data Fig. 10). This coincident encoding mechanism could help to ensure that the brain of the pupil selectively forms representations of songs produced by suitable adult tutors, and not of extraneous auditory stimuli. Although DA-dependent modulation of auditory cortical representations has previously been linked to perceptual learning28, a notable feature of the DA-dependent process of auditory encoding described here is that it occurs in a vocal motor region and rapidly drives vocal imitation. More broadly, DA signalling is enhanced in the motor cortex of primates relative to other mammals29,30, raising the possibility that augmented DA signalling in motor regions of songbirds and primates reflects a convergent neural architecture for promoting motor imitation in response to social models.
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
Juvenile male (15–90 days old), adult male (>200 days old) and adult female (>200 days old) zebra finches (Taeniopygia guttata) were obtained from the Duke University Medical Center breeding facility. All experimental procedures were in accordance with the NIH guidelines and approved by the Duke University Medical Center Animal Care and Use Committee. Birds were kept under a 14:10-h light:dark cycle with free access to food and water. Data were collected from 96 birds (Supplementary Table).
Songs were automatically recorded with Sound Analysis Pro (SAP2011)31 in a soundproof box. Vocalizations of >10 ms were detected by thresholding of the recorded sounds. Imitation of the tutor song was quantified as percentage of similarity (asymmetrical similarity) between the song motifs from pupil birds and their tutors using SAP201131 with default parameters for zebra finches, and reported as tutor song similarity. First, the song motif (a stereotyped sequence of syllables constituting an adult zebra finch song) of each bird was determined as the most frequently observed syllable sequence. Then, the percentage of similarity was calculated for representative song motifs randomly chosen from pupils and their tutor, and averaged across ≥10 comparisons to report as tutor song similarity. For immature subsongs that do not have a stereotyped song motif, we used a randomly chosen part of the subsongs with a duration that was similar to the tutor song motif for calculating the percentage of similarity. For isolated birds in Extended Data Fig. 4c, the percentage of similarity was calculated between the song motifs from isolated birds and unrelated, normally raised adult zebra finches. A song bout was detected as successive vocalizations with ≥3 syllables (to exclude call bouts) separated by an interbout interval of >400 ms. Kurtosis of vocal duration and Wiener entropy variance were calculated based on all of the song bouts in each 90-min time window.
Tutoring of juvenile birds
Juvenile birds were raised by their parents with their siblings until around 45 days old in experiments depicted in Fig. 3a–f. Otherwise, juvenile birds were separated from their parents and siblings at 15–30 days of age (that is, tutor-naive juveniles), and encountered an unfamiliar adult male (tutor) only during tutoring sessions. During a tutoring session, a juvenile bird and tutor were separated by a plastic grating or transparent glass, so they could acoustically and visually interact but direct physical interactions were prevented. The tutor was either manually introduced into the neighbouring chamber by an experimenter, or presented through an electric glass for which the transparency could be remotely controlled. Attention of juvenile birds to the tutor was quantified as the time that juvenile birds were awake and near the tutor without foraging, drinking, preening or singing, and normalized to the total time of observation (>5 min) during tutoring sessions. Untutored isolated birds depicted in Extended Data Fig. 4b, c were kept isolated from adult males until 90 days of age.
Detailed procedures of surgery were previously provided23. In brief, juvenile birds were anaesthetized with 2% isoflurane inhalation and placed on a custom stereotaxic apparatus with a heat blanket. Target cites for injection and implantation were determined by stereotaxic coordinates and multiunit activity. Stereotaxic coordinates, measured from the bifurcation of the midsagittal sinus, were 0.0 mm rostral, 2.4 mm lateral and 0.5 mm ventral for HVC; 3.4 mm rostral, 0.5 mm lateral and 6.3 mm ventral (head angle of 58°) for PAG; 5.8 mm rostral, 1.6 mm lateral and 3.0 mm ventral (head angle of 40°) for Area X; and 1.3 mm rostral, 1.2 mm lateral and 0.5 mm ventral for the caudal mesopallium (CM). Reagents or viruses were injected using Nanoject-II (Drummond Scientific). Viral injections were performed bilaterally with a volume of 483–966 nl per hemisphere. Viruses were obtained from the Penn Vector Core (Pennsylvania, USA), UNC Vector Core (Chapel Hill, USA), Janelia Virus Service Facility (Ashburn, USA), and Vigene Biosciences (Rockville, USA). Experiments were performed >30 days after the viral injection. Birds with unsuccessful injection or implantation were discarded from the analysis.
Injection of 6-OHDA
Juvenile birds received bilateral injection of 200–450 nl 6-OHDA solution into the HVC at either around 30 days of age (mean ± s.d.: 30.1 ± 4.2 days of age; range: 25–34 days of age; n = 7) or around 45 days of age (mean ± s.d.: 44.5 ± 3.0 days of age; range: 39–47 days of age; n = 6). The solution was PBS-based and included 5–20 mM 6-OHDA hydrochloride (Santa Cruz, sc-203482), 2–10 mM l-ascorbic acid (Millipore/Sigma, A92902), and 1 μM desipramine hydrochloride (Tocris, 3067), which was included as an inhibitor for noradrenaline and serotonin transporters to protect noradrenergic and serotonergic neuron terminals at the injection site. Control birds received an injection of PBS with 2–10 mM ascorbic acid and 1 μM desipramine at around 30 days of age (mean ± s.d.: 29.3 ± 3.6 days of age; range: 22–32 days of age; n = 7). Drugs were dissolved into PBS immediately before injection in place of equimolar NaCl (working solution: around 300 mOsm, pH 7.3). After injection, birds were returned to their original home cage until approximately 45 days of age when they were isolated in a soundproof box until 90 days of age.
Microdialysis infusion of drugs
Tutor-naive juveniles (around 45 days of age; mean ± s.d.: 43.8 ± 5.5 days of age; range: 32–57 days of age; n = 34) received bilateral implantation of a microdialysis probe. After 1–3 days of implantation (mean ± s.d.: 45.5 ± 5.8 days of age; range: 33–60 days of age; n = 34), tutoring sessions were conducted for five consecutive days. Each tutoring session consisted of 90-min tutor presentation. Drug was infused into the target area (HVC, Area X, CM or PAG) either 90 min before or immediately after the tutor presentation, and washed with saline 180 min after the injection (Fig. 3g). The tutor bird typically sang >30 motifs in a session (see Extended Data Fig. 5e). For a session in which the tutor did not sing any song, an additional tutoring session was conducted on the next day. As a blocker for D1- and D2-type receptors, 5 mM R(+)-SCH-23390 hydrochloride (Millipore/Sigma, D054) and 5 mM S-(−)-sulpiride (Tocris, 0895) were respectively used and dissolved into saline. To inactivate PAG, 2.5 mM muscimol (Millipore/Sigma, M-1523) dissolved into saline was infused into the PAG.
Histology and imaging
Birds were deeply anaesthetized with intramuscular injection of 20 μl euthasol (Virbac) and transcardially perfused with PBS, followed by perfusion with 4% (wt/vol) paraformaldehyde (PFA) in PBS. The removed brain was post-fixed and cryoprotected with 30% (wt/vol) sucrose and 4% (wt/vol) PFA in PBS overnight. Frozen sagittal sections (thickness of 50 μm) were prepared with a sledge microtome (Reichert) and collected in PBS. For immunohistochemistry, sections were washed twice in PBS, permeabilized with 0.3% Triton X-100 in PBS (PBST) for 1 h, blocked with 10% Blocking One Histo (06349-64, Nacalai Tesque) in PBST for 1 h, and incubated with rabbit primary antibody against TH (1:500, AB152; Millipore/Sigma) or rabbit primary antibody against dopamine beta-hydroxylase (DBH) (1:2,000, 22806; ImmunoStar) in PBST with 10% Blocking One Histo at 4 °C overnight. Then, sections were washed three times in PBST and incubated with anti-rabbit secondary antibody (1:500; Jackson ImmunoResearch) in PBST at room temperature for 1 h, followed by three washes in PBS. Sections were coverslipped with Fluoromount-G (SouthernBiotech), and then imaged with a confocal microscope (SP8; Leica) through a 20× objective lens controlled by LAS X software (Leica). To label PAG neurons that project to the HVC or Area X, dextran Alexa Fluor 488 (D-22910; ThermoFisher) was injected into the HVC (age: mean ± s.d.: 35.3 ± 7.0 days of age; range: 28–42 days of age; n = 3) or Area X (age: mean ± sd: 47.7 ± 15.3 days of age; range: 36–65 days of age; n = 3) of juvenile birds 4–7 days before perfusion. Retrogradely labelled neurons were manually counted in PAG and VTA/SNc, each of which was densely packed with TH+ neurons. Images are shown as maximum-projected images of sagittal sections. To quantify TH+ fibres in the HVC, TH+ fibres in HVC shelf/nidopallium caudolateral (NCL), and DBH+ fibres in the HVC, the fibre density was calculated in >0.04 mm2 areas from each region as the fraction of areas with fluorescence higher than mean + 10 s.d. of the background fluorescence. For analysis of HVC shelf/NCL, a >0.04 mm2 region located approximately 0.6 mm ventral of the HVC was manually selected.
Two-photon imaging and analysis
Viruses encoding DA sensors (AAV2/9-hSyn-GRABDA1h or AAV2/9-CAG-GRABDA1h), developed in the Y.L. laboratory19, were injected into the HVC of tutor-naive juveniles (approximately 30 days old, mean ± s.d.: 32.6 ± 5.3 days of age; range: 25–39 days of age; n = 5), and the HVC was imaged after implantation of a head-post and cranial window >30 days later (mean ± s.d.: 66.6 ± 6.0 days of age; range: 60–73 days of age; n = 5). To ablate DA-releasing PAG neurons, 200 nl 6-OHDA solution (10 mM 6-OHDA, 10 mM l-ascorbic acid, and 1 μM desipramine hydrochloride) was injected into PAG two days before imaging. Images were collected at 15.5 Hz with a resonant-scanning two-photon microscope (Neurolabware) that applies a mode-locked titanium sapphire laser (Mai Tai DeepSee) at 920 nm through a 16× objective lens (0.8 NA water immersion, Nikon). The objective lens was covered with black cloth to prevent room light from being detected by the photomultipliers. During imaging, a head-fixed bird in a dim room experienced playback of an adult zebra finch (tutor) song bout (3 s; seven introductory notes and three motifs comprising five syllables), encounters with an adult male tutor, encounters with an adult female bird, and a singing tutor with a randomized order. Images were acquired >10 trials for each condition, and regions of interest (ROIs) were automatically or manually selected after image alignment with MATLAB programs (Scanbox). After subtraction of background fluorescence in an annular region surrounding each ROI, signals were calculated as mean fluorescence within each ROI. Then, the ΔF/F of the ROI (%) was calculated for each trial as 100 × (F(t) − F0)/F0, where F(t) was a time series of ROI signals, and F0 was the average of baseline ROI signals for the 5-s period just before the onset of stimulus presentation. Mean ΔF/F was calculated for the 5-s period after the onset of stimulus presentation, and averaged across trials in each condition.
Tutor-naive juvenile birds received an injection of AAV2/9-CAG-ChR2-mCherry, AAV2/1-CAG-ChR2-mCherry or AAV2/9-CAG-NRX-ChR2-YFP into the PAG at around 35 days of age (mean ± s.d.: 34.0 ± 4.8 days of age; range: 30–40 days of age; n = 9). A laser was bilaterally applied through optic fibres (core: 200 μm; Thorlabs) implanted into the HVC. Juvenile birds received a tutoring session per day for five consecutive days starting at around 60–70 days of age (mean ± s.d.: 64.0 ± 4.9 days of age; range: 61–71 days of age; n = 9). In each tutoring session, a juvenile bird experienced playback of a song bout (mean amplitude: 70 dB sound pressure level, seven introductory notes and three motifs comprising five syllables) 10 times (30 motifs) within 30 min. To block DA signalling in the HVC, DA blockers were infused into the HVC with microdialysis probes 90 min before the tutoring session, and washed with saline immediately after the tutoring session (n = 3). Experimental birds received repetitive laser stimulation (10 ms; 20 Hz) throughout the playback. Control birds consisted of a group that received an injection of viruses encoding GFP and implantation of optic fibres (n = 2, scAAV2/9-CMV-GFP or AAV2/9-CAG-GFP) at around 35 days of age (mean ± s.d.: 36.5 ± 6.4 days of age; range: 32–41 days of age; n = 2), a group that did not receive viral injection but implantation of optic fibres (n = 2), and a group that did not receive injection, implantation or laser stimulation (n = 2). These groups listened to playback in the same way as experimental birds (age: mean ± s.d.: 58.5 ± 8.5 days of age; range: 54–73 days of age; n = 6), and were analysed together since we did not find any significant differences in learning abilities between these groups.
Chronic recording from the PAG and HVC
Tetrodes (A2x2-tet-3/10mm-150-150-121, NeuroNexus) were implanted into the HVC or the PAG of tutor-naive juveniles (age: mean ± s.d.: 51.3 ± 13.4 days of age; range: 27–71 days of age; n = 11). Birds were habituated to a dummy probe (1.5–2 g) on the head for approximately seven days before the implantation. Data were collected with a universal serial bus (USB) interface board (RHD2000; Intan Technologies) after band-pass filtering (0.2–10 kHz) and sampling at 30 kHz with a small amplifier board (RHD2132 16-Channel; Intan Technologies) on the head of the bird. Unit activity was sorted in a semi-automated fashion with custom C++ software using a support vector machine algorithm (M.T.). Unit activity with a mean amplitude >3 s.d. of noise was used for subsequent analyses. Recording of song-related activity was triggered by xpctarget in MATLAB (MathWorks). To block DA signalling in the HVC, juvenile birds received an injection of 6-OHDA into the HVC 2–5 days before tetrode recording from the same HVC. The mean firing rate of PAG neurons was calculated for >10 trials with >0.5 s after the onset of singing or song playback and 5 s after presentation of a male or female bird, and averaged after normalization with mean spontaneous firing rate calculated for >10 s before the presentation of stimuli. Probability of burst activity in HVC neurons was calculated for >300 s spontaneous activity before and after exposure to a live tutor. Coefficients of variance of the firing rate across trials of HVC neurons were calculated for 50-ms bins with a hop size 1 ms across >15 trials, and reported as the mean of the coefficients of variance of the firing rate from all the bins in the motif (>0.5 s) if the mean firing rate during playback was >0.05 Hz. For data analysis, Igor Pro (WaveMetrics), MATLAB and Microsoft Excel were used.
Data are shown as mean ± s.e.m., unless otherwise noted. Two-way ANOVAs were performed in MATLAB to examine the significance of the main effect of 6-OHDA (F2,85 = 53.10, P < 0.001; Fig. 3e–f), DA blockers on the HVC, DA blockers on the CM and muscimol on PAG (F5,99 = 23.17, P < 0.001; Fig. 3h and Extended Data Fig. 5c, h, k), DA blockers on Area X (F1,30 = 0.22, P = 0.640; Extended Data Fig. 6c), optogenetic activation of PAG terminals in the HVC (F2,47 = 16.61, P < 0.001; Fig. 3j and Extended Data Fig. 7f), followed by a post hoc Tukey–Kramer test to report significant differences between conditions at each age window. To examine the different proportion of labelled neurons in the PAG and VTA/SNc, χ2 tests were performed. Two-way ANOVAs were performed in MATLAB to examine significance of the main effect of blockage of DA signalling on kurtosis syllable duration (F1,39 = 19.69, P < 0.001; Fig. 4h), entropy variance (F1,39 = 4.84, P = 0.034; Fig. 4i) and song rate (F1,39 = 0.16, P = 0.691; Extended Data Fig. 9), followed by a Tukey–Kramer test to report significant differences between conditions at each time window and by a Student’s two-sided paired t-test with Bonferroni correction to report significant differences between before and after exposure to tutor songs. One-way ANOVAs were performed in MATLAB to examine the main effect of different conditions in Fig. 1k and Extended Data Fig. 2b (F4,93 = 6.84, P < 0.001), Fig. 2i (F4,23 = 10.31, P < 0.001), Extended Data Fig. 3c (F2,12 = 13.42, P < 0.001), Extended Data Fig. 3d (F2,12 = 0.14, P = 0.870), Extended Data Fig. 4a (F2,17 = 0.28, P = 0.757), Extended Data Fig. 5d (F2,7 = 30.40, P < 0.001), and Extended Data Fig. 5e (F2,10 = 0.78, P = 0.486), each followed by a Tukey–Kramer test to report significant differences between conditions. In other analyses, Student’s paired t-tests (Figs. 1k, 2i, 4h, i and Extended Data Figs. 2b, 8d–f) or Student’s unpaired t-tests (Extended Data Figs. 3e, 4c) were performed in Microsoft Excel. Multiple data from a bird are indicated with the same markers in Fig. 1c, g, k, 2i, 4e, f and Extended Data Figs. 1b, c, e–g, 2b, 3c–e, 8d–f. Statistical tests were performed two-sided. Asterisks show P < 0.050.
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Custom code or software is available from the corresponding author upon reasonable request.
The datasets generated and analysed during the current study are available from the corresponding author upon reasonable request.
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We thank J. Hatfield for constructing AAV2/9-CAG-GRABDA1h; S. Nowicki, S. Peters, C. Sturdy, F. Wang and S. Soderling for critical discussion and for reading earlier versions of this manuscript. This work was supported by JSPS Postdoctoral Fellowship for Research Abroad (M.T.), the National Basic Research Program of China 973 Program Grant 2015CB856402 (Y.L.), the American BRAIN Initiative project 1U01NS103558-01 (Y.L.), NIH Grant 1R01-NS-099288 (R.M.) and NSF IOS-1354962 (R.M.).
Nature thanks O. Tchernichovski, L. Zweifel and the other anonymous reviewer(s) for their contribution to the peer review of this work.