Ex vivo cAMP stimulation and measurement of cAMP levels using ELISA

Fresh habenula and IPn samples were micro-dissected from Tcf7l2^WT and Tcf7l2^mut rats. The tissue samples from two animals were pooled together for one experimental replicate. A total of 3 experimental replicates per condition were used. Tissue was lightly homogenized with a motorized tissue grinder in 40 μl phosphate buffered saline. To stimulate cAMP production in tissue homogenates, 5 μl of vehicle (PBS) or 1 mg/ml Ex-4 (final concentration 100 μM) was spiked into the tube. Samples were briefly mixed and incubated for 30 min at 30°C. 5 μl of 1 M HCl was added to stop the reaction and lyse the tissue. Samples were stored at −80°C until use. When thawed, samples were centrifuged at 13,000 x g for 10 min at 4°C. The concentration of cAMP in the supernatant obtained from the rat habenula and IPN extracts was measured using the cAMP Direct Immunoassay Kit (Biovision Inc, Milpitas, CA, USA) per the manufacturer’s instructions.