Cm300

The brains were prepared using the same method as in histology and cut into 40-µm-thick coronal sections with a cryotome (CM300, Leica). The sectioned brain regions encompassed the habenula and the RMTg. Brain sections were incubated in primary antibodies for 1 h at 37 °C. The primary antibodies were diluted in PBS containing 3% bovine serum albumin (BSA) and 0.2% Triton X-100. The following primary antibodies were used: anti-c-Fos antibody (1:500; #SC-52G, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-GABA antibody (1:500; #A2052, Sigma-Aldrich, St. Louis, MO, USA). Next, the sections were washed with PBS and incubated in a cocktail of Alexa Fluor® 488 donkey anti-goat (1:500; #A-11055, Thermo Fisher Scientific) or Alexa Fluor® 647 donkey anti-rabbit (1:500; #711-605-152, Jackson Immuno Research Inc., West Grove, PA, USA) conjugated secondary antibodies in PBS containing 3% BSA and 0.2% Triton X-100 for 2 h at room temperature. The secondary antibody was washed with PBS and further incubated with Hoechst (1:1000; #H3570, Thermo Fisher Scientific) at room temperature for 10 min. The sections were immersed in mounting solution and images were captured using a TCS SP8 dichroic/CS microscope (Leica).

Mice were anaesthetized with a mixture alfaxalone (40 mg per kg): xylazine (10 mg per kg) and perfused with 0.9% saline and then with 4% paraformaldehyde. The brains were removed from the skulls and postfixed in 4% paraformaldehyde overnight at 4 °C. After postfixation, the brains were incubated in 30% sucrose at 4 °C. The brains were cut into 100-µm-thick coronal sections with a cryotome (CM300, Leica); sectioned brain regions encompassed the habenula. Next, sections were washed with phosphate-buffered saline (PBS) three times for 5 min at room temperature. The washed sections were incubated with Hoechst 33342 (H3570, Thermo Fisher Scientific), a blue fluorescent stain that binds double-stranded DNA, at room temperature for 10 min. The stained sections were immersed in mounting solution for 30 min at 37 °C. The sections were viewed and photographed using a TCS SP8 dichroic/CS microscope (Leica).