Fig. 1. Multi-Region Neuropixels Recordings in Awake-Singing Songbirds.

a. Illustration of the experimental objective to record neural activity alongside the song of freely-singing zebra finches. b. A single Neuropixels probe is implanted chronically at the required angle to simultaneously record the extracellular activity of HVC and RA neural populations. c. Example histological sample for confirmation of recording location, showing electrode track (fluorescent trace) through a saggittal section of the caudal telencephalon with both HVC and RA visible. d. Amplitude waveform and corresponding spectrogram of a recorded song bout (top). Below, spike-trains from spike-sorted single-unit activity (SUA) and multi-unit activity (MUA) clusters in the HVC and RA regions are time-aligned with the audio recording. On the left, a sketch of the Neuropixels probe illustrates the probe sites covering each brain region, estimated from the probe’s geometry, brain histology, and observed modulated neural activity. e. Amplitude waveform and spectrogram of an example motif within the bout (top). Below, example raster plots and PSTHs of one putative HVC projection neuron, one putative HVC interneuron, and one putative RA neuron across 10 renditions of the same motif and aligned to the onset of the first syllable in the motif.