Novel Object Recognition Testing

We used a black Plexiglas® NOR chamber (52X52X30cms) as described for juvenile rats (P29-40) (Reger et al., 2009) housed in a small room with soft lighting. Objects used for testing were small, plastic and easily cleaned objects secured to the box using Velcro ®. Interaction of the rat with an object was measured using automated video tracking and data analysis equipment from EthoVision® vXT 13 (Noldus Information Technology, Wageningen NL) and confirmed by dual observer analysis of the videotaped session when needed. Exploration was timed for the duration that the rat’s nose was within 2 cms of the object, as determined by a digitally drawn arena. We did not consider time spent rearing, if present, as exploration. We tested rat pups (n=5 per group) at PID 14 (age P31). We cleaned the box and objects between rat pups with 70% alcohol to remove odor traces. Rat pups were acclimated to handling prior to commencing NOR procedures. During the Habituation phase, each rat pup individually explored the empty box for five minutes on day one and fifteen minutes on day two to minimize stress and ensure acclimation to the testing environment. During the Familiarization phase, we placed a single rat in the arena containing two identical objects for five minutes. We exposed half of the rats to two identical objects in one configuration (see black spheres in Fig. 1B) and the other half to the mirror image configuration (see black spheres in Fig 1B). Each rat returned to the arena 24 hours later for Novel testing over five minutes. Each rat was exposed to one object from the familiarization trial (black sphere) and to one novel object (red triangle) in the configurations shown in Fig 1B, alternating between A1 and A2 for every rat exposed to Familiarization A1 and between B1 and B2 for every rat exposed to Familiarization B. Performance on the testing phase was measured by the percent novel exploration time (100* (N/ (N+F)) where N is the time spent on the Novel object and F on the familiar. A higher exploration time is considered to reflect greater memory ability, while an exploration time of 50% is not better than chance.