Lhx8 regulates TrkA expression and cholinergic function in vivo. AAV-GFP or AAV-HA-Lhx8 was injected into the medial septum of 8-week-old rats. One month after AAV injection, the rats were sacrificed. Frozen sections of brain were prepared for immunohistochemical detection of TrkA (A and B) and ChAT (A and C), and tissue extracts of medial septum were used for immunoblotting (D–F). A, bright field microscopy revealed more intense brown 3,3′-diaminobenzidine staining for TrkA and ChAT in the medial septum of rats injected with AAV-HA-Lhx8 than AAV-GFP-infected ones. Scale bar, 300 μm. B and C, no significant difference in the number of TrkA- and ChAT-positive cholinergic neurons in a section with the same coordinate was observed. D, protein extracts from AAV-GFP- or AAV-Lhx8-injected medial septums were immunoblotted with an antibody directed against TrkA, ChAT, Lhx8, GFP, HA, or actin. E and F, level of TrkA and ChAT protein was normalized to that of actin and expressed as a percentage of the value of AAV-GFP-injected medial septum. Data are presented as the mean ± S.E. n = 5; **, p < 0.01; Student's t test in E. n = 5; *, p < 0.05; Student's t test in F.