Figure 2

Effects of CHAT knockdown in medial habenula on depression-like behaviors. (a) Schematic representation of the AAV vector engineered to induce CHAT knockdown. (b) Experimental paradigm for behavioral testing of rats infected with the virus. (c) Representative photomicrographs of Hb slices from the rats injected with AAV-sh-SCR (control) or AAV-sh-CHAT (CHAT knockdown). CHAT protein expression was visualized as red immunofluorescence. Cell nuclei and viral infection were visualized using Hoechst staining (blue) and GFP (green), respectively. The inset box shows a higher magnification of an infected MHb region. None of the neurons infected with the AAV-sh-CHAT showed CHAT expression. Scale bar, 50 μm. (d) VACHT expression is not affected by AAV-sh-CHAT. Hb neurons injected with AAV-sh-CHAT were immunostained for VACHT (red) and EGFP (green). Scale bar, 50 μm. (e) CHAT and VACHT western blots from the Hb of rats injected with AAV-sh-CHAT. Rats were injected with AAV-sh-SCR (control) or AAV-sh-CHAT, which were expressed for 3 weeks before western blot analysis. (f) Quantification of the effects of shRNA-mediated CHAT knockdown. Data represent mean ± SEM (sh-SCR, n = 4 rats; sh-CHAT, n = 4; *P < 0.05, Student’s t-test). (g) VACHT expression is not affected by AAV-sh-CHAT. Data represent mean ± SEM (sh-SCR, n = 4 rats; sh-CHAT, n = 4) (h–j) Behavioral effects of expressing AAV-sh-CHAT in the MHb on sucrose preference (h), total fluid intake during SPT test (i), and forced swim (j). Data represent mean ± SEM (sh-SCR, n = 20; sh-CHAT, n = 27; **P < 0.01, Student t-test).