Figure 2. HVC_Av neurons transmit motor-related signals during song production.

a) Schematic of imaging experiments employing a miniature epifluorescence microscope (Inscopix) for in vivo calcium imaging of HVC_Av neurons expressing GCaMP6s. b) Bulk fluorescent signal measured in HVC at rest (top left) and during singing (top right). Bottom panel shows bulk change in fluorescent signal during singing of multiple song bouts (song onset to song offset for each bout indicated by blue shading). Scale bars, 100 μm x 100 μm (top panels) and 1% and 2s (bottom panel). c) Average change in bulk fluorescence signals measured during singing in birds expressing GCaMP6s in HVC_Av neurons with their hearing intact (top panel, n=4) and after deafening (middle panel, n=2), and in a bird not injected with GCaMP (bottom panel, n=1). Onset and offset of song bout indicated by blue shading. Scale bars, 2% and 1s. d) Example field of GCaMP-labelled HVC_Av neurons imaged through miniature microscope in a singing zebra finch. Putative individual cells, which were identified with CNMF analysis, are indicated by colored outlines. Scale bars, 100 μm x 100 μm. e) Activity of five putative HVC_Av neurons indicated in panel d) during a single song bout comprising two motifs (sound spectrogram at top; color in the sound spectrogram indicates relative power with black indicating the lowest power (during silent intervals) and red indicating the highest relative power within the vocal range). f) Singing related activity of five different putative HVC_Av neurons from the same bird from which d) and e) were collected but several days after deafening; activity is aligned to a single bout comprising two motifs. Scale bars in e) and f), 20 arbitrary activity units and 500 ms.