(A) Schematic overview of the song system. HVC, proper name; RA, robust nucleus of the arcopallium; LMAN, lateral magnocellular nucleus of the nidopallium; Av, avalanche; DLM, medial portion of the dorsolateral thalamic nucleus; D, dorsal; P, posterior. (B) Circuit diagram of the song system. Arrowheads and closed circles indicate excitatory and inhibitory connections, respectively. NC, caudal nidopallium; Arco., arcopallium; NR, rostral nidopallium; Stri., striatum. (C) Schematic of SLCR-seq. Fresh-frozen brains were cryosectioned for Laser Capture Microdissection (LCM). Individual sections of regions of interest were collected into wells of 96-well plates, and then total RNA was purified using an optimized solid phase reversible immobilization (SPRI) protocol. After RNA purification, 3’-end sequencing libraries were prepared containing unique molecular identifiers (UMI) using a custom protocol. (D) Left: Experimental overview of SLCR-seq on hearing and deaf birds. After a baseline period of song recording, birds were either deafened through bilateral cochlear removal or underwent a sham surgery. After 4, 9, or 14 days post-surgery, birds were euthanized and SLCR-seq libraries were prepared from HVC, NC, RA, Arco., LMAN, NR, Area X, and Stri. Right: Uniform Manifold Approximation and Projection (UMAP) plot of SLCR-seq data colored by section position. Each point reflects the gene expression profile of a single SLCR-seq sample. Samples show segregation by broad anatomical area — striatal (Area X), nidopallial (HVC, NC, LMAN, NR), arcopallial (RA, Arco.) — and song system nuclei from surrounding areas. (E) Normalized log gene expression data of three example genes — SLC17A6, PVALB, and AR. Each point is gene expression in a single SLCR-seq sample. SLC17A6 is a marker for glutamatergic cells and is distinctly depleted in the striatal samples; PVALB and AR are two genes known to be enriched in song system nuclei. (F) Coronal anatomical atlas representation of the expression of the three genes shown in panel (F). Each region is colored according to the log gene expression value. D, dorsal; L, lateral.