Extended Data Fig. 4: Calcium indicator is expressed exclusively in HVC excitatory neurons and imaged in annotated ROIs.

a, Sagittal slice of HVC showing GCaMP-expressing PNs (experiment repeated in five birds with similar results). b, We observed no overlap between transduced GCaMP6f-expressing neurons and neurons stained for the inhibitory neurons markers calretinin (CR), calbindin, and parvalbumin (calretinin stain shown, staining experiment repeated six times for each marker with similar results). c–e, Examples of daily ROI annotation in three birds (1–3). Coloured circles mark different ROIs, manually annotated on maximum fluorescence projection images on an exemplary day (see Methods). f, Maximum fluorescence images (from bird 1; see Methods) revealing fluorescence sources, including sparsely active cells, in the imaging window across multiple days.