Cognitive and affective characterization

Object location memory (OLM) and object recognition memory (ORM) testing occurred following OF and EPM testing (mean ± SD: OLM: dKA = 71 ± 27 d postinjection; vKA = 81 ± 27 d postinjection; saline = 66 ± 17 d postinjection; ORM: dKA = 74 ± 16 d postinjection; vKA = 77 ± 29 d postinjection; saline = 63 ± 14 d postinjection). The order of the two tests was alternated between subjects, and there was 1 week between the end of one testing session and the beginning of habituation for the next memory test. The design of the tests was based on previously published protocols (Leger et al., 2013; Vogel Ciernia and Wood, 2014). Mice were handled for 5 d before beginning habituation, once a day for 90 s. After handling, mice were habituated to the OLM or ORM arena twice a day for 5 min at a time, for 3 consecutive days. The following day, two identical objects were introduced to the arena. Subjects were placed into the arena and allowed to explore for 10 min. The following day, two objects were again put into the arenas: for the OLM, object identity remained the same from the previous day, but the location of one object was moved. For the ORM, both objects were put in the same location as the previous day, while one object was changed to a novel object. Subjects were allowed to explore for 5 min. The OLM arena was a rectangular [43 × 22 × 20 cm (length × width × height)] arena painted black, with a white stripe 4 cm in diameter at the center of a long side. The ORM arena was a circular (diameter, 30 cm; height, 26 cm) white arena with a 4 cm black stripe. A thin layer of bedding material covered the floor of the ORM arena. Training and testing sessions were recorded and manually analyzed off-line for bouts of object exploration. A discrimination index (DI) was calculated as follows: difference in time spent investigating novel and familiar objects divided by the sum of all object exploration time. Investigation was defined as the orientation of the subject’s nose directly toward the object and within 1 cm of the edge of the object, exclusive of active engagement in a different activity (e.g., digging). To ensure the consistency of scoring, analysis was performed by two experimenters blinded to the experimental condition. A pre-established exclusion criteria was set at <3 s of total object exploration during the testing session or biased performance during the training session, defined as the absolute value of the DI (using an arbitrary novel object designation) >20. In total, 5 ORM subjects (1 dKA, 1 vKA, 2 saline) and 10 OLM subjects (4 dKA, 4 vKA, 2 saline) were excluded. After finishing the first memory test, subjects were housed in a custom cage for the sucrose consumption task (SCT; described below) before being returned to standard housing for the second memory test.

SCT testing occurred on average 69 d postinjection (mean ± SD: dKA = 74 ± 23 d postinjection; vKA = 82 ± 26 d postinjection; saline = 63 ± 13 d postinjection). Following a published protocol (Katz, 1982; Klein et al., 2015), animals had access to two identical water bottles in a custom cage. After habituating the animal to the cage and water bottles for 3 d, both water bottles were refilled: one with drinking water and the other with a 4% sucrose water solution. Approximately 24 h later, the bottles were both replaced with drinking water. This pattern was repeated for a total of 2 d with access only to drinking water, and 2 d with access to a sucrose solution. The side of the sucrose-containing bottle was counterbalanced across days. Before refilling, bottles were weighed to analyze consumption from the previous day. After refilling, bottles were weighed to set a baseline for the next day. Collection and analysis of SCT data were performed blinded to experimental condition.