a) Schematic showing the design of retrograde labeling experiments. Different fluorescent retrograde tracers were injected into Av (dextran, Alexa Fluor 594), RA (dextran, Alexa Fluor 488), and Area X (fast blue). b) Parasagittal section through retrogradely labeled HVC (left panel) reveals that neurons projecting into Avalanche (red) are distinct from neurons projecting to Area X (blue) or RA (green). Inset shows magnified image outlined by white box. c) Images of individual HVCAv neurons retrogradely labeled by AAV2/9 GFP injection into Av. d) Visualized whole-cell current clamp recordings from retrogradely-labeled HVC projection neurons in brain slices in response to 500ms current injection (−50 and +200 pA; dashed line = −80mV). HVCAv neurons differ from HVCX neurons in their intrinsic properties, including their e) spike adaptation rate (two-sample t(10) = 3.5, P = 0.005 for HVCAv neurons (n = 5) versus HVCX neurons (n = 9); two-sample t(9) = 2.6, P = 0.02 for HVCX neurons (n = 9) versus HVCRA neurons (n = 6)), f) spike gain (two-sample t(10) = 3.3, P = 0.007 for HVCAv neurons (n = 7) versus HVCX neurons (n = 9); two-sample t(6) = 2.7, P = 0.03 for HVCX neurons (n = 9) versus HVCRA neurons (n = 7)), g) input resistance (two-sample t(10) = 2.3, P = 0.04 for HVCAv neurons (n = 7) versus HVCX neurons (n = 9)). h) HVCAv neurons differ from HVCRA neurons in their Ih amplitude (two-sample t(7) = 2.5, P = 0.04 for HVCAv neurons (n = 7) versus HVCRA neurons (n = 7); two-sample t(9) = 3.2, P = 0.01 for HVCX neurons (n = 9) versus HVCRA neurons (n = 7)). HVCAv neurons differ from HVCX and HVCRA neurons in i) the coefficient of variation of their DC-evoked interspike intervals (two-sample t(5) = 2.9, P = 0.03 for HVCAv neurons (n = 7) and HVCRA neurons (n = 6); two-sample t(12) = 3.0, P = 0.01 for HVCAv neurons (n = 7) and HVCX neurons (n = 9)). Scale bar, 100μm in B (left panel) and 15μm in B (right panel). Scale bar, 100μm in B and 15μm in C.