Tail Suspension Test (TST)

The TST was employed to evaluate depression-like behavior and was conducted as previously described (Can et al., 2012). Mice were suspended by their tails from one half of a conditioned-place preference (CPP) chamber constructed from clear acrylic (20 cm length × 20 cm depth × 20 cm height) and placed on its side, which was nested within a white acrylic OF box (40.6 cm length × 40.6 cm width × 30 cm height) placed on its side. The CPP–OF box setup was used to improve video contrast and facilitate mouse movement tracking. The CPP–OF apparatus was positioned at the edge of the lab bench (3 ft height) in the procedure room, such that when suspended by their tails, mice dangled over the side of the lab bench. Before the experiment, a hollow cylindrical tube used to ensheath the tail to impede tail climbing behavior once suspended and a piece of tape used to attach the tail to the CPP box were prepared for each experimental mouse. Specifically, a Falcon 3 ml Transfer Pipet (Corning Inc.) that had the bulb and tip cut off was trimmed down to a 4-cm long hollow cylindrical tube and a 13-cm long piece of VWR General-Purpose Laboratory Labeling Tape (12.7 mm width; VWR International, LLC) was marked with a permanent marker at 5.5 cm, 10 cm, and 11 cm relative to one end. To carry out the experiment, a mouse’s tail was passed through the hollow tube and the section of prepared tape between 10–11 cm was wrapped around the end of a mouse’s tail leaving 2–3 mm of the tail exposed at the end. Experimental mice were transported from their home cage in the palm of the experimenter’s hand and the tape position marked at 5.5 cm was attached to the top of the CPP box in the center, such that mice could not contact the sides of the CPP box. Mice were then suspended by their tails for the 6-min trial. The CPP–OF apparatus was cleaned with 70% ethanol and paper towels in between trials. Mouse movement was recorded with a Canon ZR90 camcorder affixed to a tripod to capture side-on immobility. Immobility was analyzed using EthoVision (version 3.0; Noldus Information Technology, RRID:SCR_004074) with an immobility threshold of 10%.