Background

The Barnes maze (Barnes, 1979) is a widely acknowledged dry alternative to the Morris Water Maze (MWM) (Morris, 1984). The Barnes maze uses bright light as a motivating factor to cause animals to locate a darkened box hidden beneath one of several escape holes using spatial cues. It has recently been shown that a Barnes maze adapted for juvenile rats can be used to assess the maturation of spatial learning and memory, as spatial navigation ability first emerges around the end of the third postnatal week (McHail et al., 2018). Unlike the MWM, the Barnes maze requires less time away from the dam and does not involve swimming and temperature stressors that can differentially impact juvenile animals (Albani et al., 2014). Also, the Barnes maze is more compatible with in vivo electrophysiological recording, enabling a greater signal to noise ratio (McHail et al., 2018).

The Barnes maze adapted for juvenile rats consists of a round white platform that is smaller than the adult maze with eight evenly spaced holes around the perimeter, a reduced number of perimeter holes compared to the adult apparatus. Distal visual cues (extra-maze contextual cues) are placed around the maze and used by the animal to determine the spatial location of an escape box hidden underneath one of the perimeter holes. These spatial cues are enlarged to allow better visualization for the younger animals (McHail et al., 2018).

Behavioral testing consists of four days: an exploration trial on Day 1, 5 blocks of training trials followed by a spatial learning probe on Days 2 and 3, and a final spatial memory probe approximately 24 h later (24 HP). The exploration trial on Day 1 allows the animal to freely roam the platform in dim lighting (no escape box) and permits the experimenter to map place cells in animals having tetrodes implanted. Soy-based infant formula droplets are scattered on the maze to encourage exploration. Training on Days 2 and 3 are conducted under bright lighting with the escape box positioned under the same escape hole for all training trials. Each block contains four trials where the animal has a maximum of 60 s to locate the goal starting from four different start locations that are randomly ordered within a block. Training blocks are separated by 15-30 min. Following the final block on each training day, a probe is conducted wherein the escape box is removed and the animal is observed for 60 s. Following the immediate probe on Day 3, the escape box is returned to conduct a final refresher block. The 24 HP is performed on Day 4 to test long-term spatial reference memory.

Animals are tested for the length of time it takes them to locate the escape box (escape latency), the distance traveled to reach the escape box (path length), and the maximum and mean movement speed during this path. These measures are repeated excluding trials for which the animal did not locate the goal (non-escape trials). The probe trials examine recall by calculating the amount of time spent near the escape box location (dwell time), distance from the goal at every second (similar to Gallagher et al., 1993), and nose pokes before and after the first goal nose poke, in addition to the measures calculated for training trials (escape latency, path length, and movement speed).