VTA projections to MST or Area X are segregated before the onset of vocal practice.

(A) Schematic of the experimental strategy used to label VTA projections to MST and Area X; (B) Darkfield image with expanded view of VTA, denoted by dashed white rectangle, scale bar 250 μm; (C) MST (magenta) and Area X (orange) projecting neurons in VTA in adults. Right to left: examples of a similar region in VTA from three different birds. Scale bar, 75 um. Dorsal is up; anterior is to the right. (D) Same as C except for late juveniles 60–65 dph; (E) Same as C except for early juveniles 30–35 dph. (F) Average cell counts per hemisphere of populations projecting to Area X (dark gray) and MST (white) from VTA in all three injection sites with an n of 12, 7, and 7 hemispheres respectively. Asterisks above vertical error bars for the late juveniles and adult populations show results of Tukey’s post-hoc analysis when comparing to appropriate early juvenile populations. Asterisks above horizontal brackets show results of Welch two sample t-tests between neuron populations within the same age group. (G) Percent of co-labeled cells in VTA that project to both MST and Area X in all three injection sites with an n of 12, 7, and 7 hemispheres respectively. (H) Proportion of Area X (black) and MST (gray) projecting cells over total labeled cells in the brain slice, progressing from medial to lateral. Error bars reflect standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.