Animals
The experiments described in this study were conducted using adult male zebra finches (Taeniopygia guttata; 120–500 days post-hatch). All procedures were performed in accordance with protocols approved by the Animal Care and Use Committee at UT Southwestern Medical Center.
Viral vectors
The following adeno-associated viral vectors were used in the experiments: rAAV2/9/fDIO–CBh–eGTACR1–mScarlet, rAAV2/9/CBh–Flippase, rAAV2/9/CBh–ChRmine–mScarlet, rAAV2/9/DIO–CAG–ChRmine–mScarlet, rAAV2/9/DIO–CAG–TeNT–mScarlet (Intellectual and Developmental Disabilities Research Center Neuroconnectivity Core at Baylor College of Medicine) and rAAV2/9/CMV–CRE–eGFP (Addgene). All viral vectors were aliquoted and stored at −80 °C until use.
Stereotaxic surgery
Aseptic stereotaxic surgeries were performed after birds were anaesthetized (isoflurane inhalation; 0.8%–1.5%).
Viral injections were performed using previously described procedures26,37,58. Briefly, a cocktail of adeno-associated viral vectors (rAAV/CBh–ChRmine in HVC, RA, area X or thalamus (2 µl per hemisphere); 1:2 of rAAV/CBh–FLP and rAAV/DIO–CBh–eGtACR1, respectively (1–2 µl total per hemisphere); rAAV/DIO–CAG–ChRmine in HVC or Uva (2 µl); rAAV/CMV–Cre in RA, area X or HVC (0.5–1 µl and 2 µl, respectively); rAAV/DIO–TeNT in HVC or Uva (2 µl); and rAAV/CMV–CRE in area X or HVC (2 µl), respectively) were injected (1 nl s−1) into target areas with a Nanoject III (Drummondsci) and glass capillaries. Experiments were conducted starting a minimum of 3 weeks after viral injections. Fluorophore-conjugated retrograde tracers (Dextran 10,000 MW, AlexaFluor 488, 568 and 647, Invitrogen; Fast Blue, Polysciences) were injected bilaterally into area X, RA or HVC (160 nl; 5 × 32 n, 32 nl s−1 every 30 s) (refs. 26,37,58). Electrophysiological mapping was used to determine the centres of HVC, NIf, mMAN, LMAN and RA, and area X, nucleus avalanche and Uva were identified using stereotaxic coordinates (coordinates relative to interaural zero: head angle, rostral–caudal, medial–lateral, dorsal–ventral (in mm). The stereotaxic coordinates were as follows: HVC (45°; anterior–posterior, 0; medial–lateral, ±2.4; dorsal–ventral, −0.2 to −0.6), NIf (45°; anterior–posterior, 1.75; medial–lateral, ±1.75; dorsal–ventral, −2.4 to −1.8), mMAN (20°; anterior–posterior, 5.1; medial–lateral, ±0.6; dorsal–ventral, −2.1 to −1.6), lMAN (20°; anterior–posterior, 5.1; medial–lateral, ±1.7; dorsal–ventral, −2.2 to −1.6), RA (80°; anterior–posterior, −1.5; medial–lateral, ±2.5; dorsal–ventral, −2.4 to −1.8), X (45°; anterior–posterior, 4.8; medial–lateral, ±1.6; dorsal–ventral, −3.3 to −2.7), nucleus avalanche (45°; anterior–posterior, 1.65; medial–lateral, ±2.0; dorsal–ventral, −0.9) and UVA (20°; anterior–posterior, 2.5; medial–lateral, ±1.6; dorsal–ventral, −4.8 to −4.2).
Optogenetic manipulations
For optogenetic stimulation, optic fibres (multimode 400 µm; 0.39 numerical aperture; ThorLabs) were implanted bilaterally dorsal to HVC, RA, area X or Uva using acrylic glue and dental cement. Although the 400-µm-diameter fibres may not completely cover the entirety of the areas, we estimated that the cone of light could stimulate the vast majority of the targeted neurons. After recovery, the implanted fibres were connected to optic fibres through ceramic sleeves. The fibres were connected to a rotary joint and interfaced with a 1.5-mm multimode fibre connected to a light-emitting diode box (Prizmatix). Light intensity was regulated to achieve a final output of approximately 10 mW. We used a custom software (pcaf; LabVIEW) to deliver optogenetic stimulation during song (200 ms or 1 s for HVC afferent stimulation, 10–50 ms for direct ChRmine somatic stimulation and 50–200 ms for antidromic HVC X stimulation). In many instances, our goal was to target as many moments as possible within a bird song motif. To achieve this, we targeted most of the motifs birds were producing using quasi-random light onset delays introduced through a transistor–transistor logic. This targeting strategy allows for a detailed analysis of motif-level effects but limits our ability to conduct meaningful song-bout-level analysis of the behaviour. We note that light delivery over HVC or other brain regions is not sufficient to cause truncations or other disruptions in singing behaviour because several experiments using light stimulation (light stimulation of afferent pathways into HVC or of area X neurons) have no effect on singing behaviour. Air sac recordings and analysis were performed as previously published15.
Lesion quantification
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